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Biochemistry An introduction

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Contents Articles Cells and water Biochemistry

1 1

Cells

Water

Structural Biochemistry

Nucleic acids

Nucleic acid

RNA

DNA

Proteins and amino acids

Protein

Amino acid

Properties of the twenty amino acids

Myoglobin

107

Hemoglobin

112

Enzyme mechanisms

129

Enzyme catalysis

129

Enzyme kinetics

137

Enzyme kinetics

137

Lipids and membranes

151

Lipid

151

Biological membrane

159

Membrane protein

161

Cell membrane

165

Carbohydrate structure

172

Carbohydrate

172

Polysaccharide

178

Intermediary metabolism

184

Metabolism

185

Overview of metabolism

185

Carbohydrate metabolism

200

Glycolysis

200

Gluconeogenesis

214

Glycogen

218

Pentose phosphate pathway

222

Citric acid cycle

226

Citric acid cycle

226

Oxidative phosphorylation Oxidative phosphorylation

233 233

Photosynthesis

245

Photosynthesis

245

Lipid metabolism

259

Fatty acid synthesis

259

Lipogenesis

267

Acetyl-CoA carboxylase

269

Fatty acid degradation

276

Beta oxidation

278

Nitrogen metabolism

283

Nitrogen fixation

283

Amino acid synthesis

288

Nucleotide

295

Urea cycle

301

Integration of metabolism

305

Hormone

305

Signal transduction

309

Diabetes mellitus

316

Informational Macromolecules

327

DNA synthesis and repair

328

DNA replication

328

DNA repair

335

Oncogenes

346

RNA synthesis and processing

350

Transcription

350

Regulation of gene expression

356

Protein synthesis and modifications

362

Translation

362

Posttranslational modification

366

Proteolysis

370

Proteasome

375

References Article Sources and Contributors

386

Image Sources, Licenses and Contributors

398

Article Licenses License

406

Cells and water Biochemistry Biochemistry, sometimes called biological chemistry, is the study of chemical processes within, and relating to, living organisms.[1] By controlling information flow through biochemical signaling and the flow of chemical energy through metabolism, biochemical processes give rise to the complexity of life. Over the last 40 years biochemistry has become so successful at explaining living processes that now almost all areas of the life sciences from botany to medicine are engaged in biochemical research.[2] Today the main focus of pure biochemistry is in understanding how biological molecules give rise to the processes that occur within living cells, which in turn relates greatly to the study and understanding of whole organisms. Biochemistry is closely related to molecular biology, the study of the molecular mechanisms by which genetic information encoded in DNA is able to result in the processes of life. Depending on the exact definition of the terms used, molecular biology can be thought of as a branch of biochemistry, or biochemistry as a tool with which to investigate and study molecular biology. Much of biochemistry deals with the structures, functions and interactions of biological macromolecules, such as proteins, nucleic acids, carbohydrates and lipids, which provide the structure of cells and perform many of the functions associated with life. The chemistry of the cell also depends on the reactions of smaller molecules and ions. These can be inorganic, for example water and metal ions, or organic, for example the amino acids which are used to synthesise proteins. The mechanisms by which cells harness energy from their environment via chemical reactions are known as metabolism.

History It once was generally believed that life and its materials had some essential property or substance distinct from any found in non-living matter, and it was thought that only living beings could produce the molecules of life.[citation needed] Then, in 1828, Friedrich Wöhler published a paper on the synthesis of urea, proving that organic compounds can be created artificially.[] The dawn of biochemistry may have been the discovery of the first enzyme, diastase (today called amylase), in 1833 by Anselme Payen.[3] Eduard Buchner contributed the first demonstration of a complex biochemical process outside of a cell in 1896: alcoholic fermentation in cell extracts of yeast.[4] Although the term "biochemistry" seems to have been first used in 1882, it is generally accepted that the formal coinage of biochemistry Gerty Cori and Carl Cori jointly won the occurred in 1903 by Carl Neuberg, a German chemist.[] Since then, Nobel Prize in 1947 for their discovery of biochemistry has advanced, especially since the mid-20th century, with the the Cori cycle at RPMI. development of new techniques such as chromatography, X-ray diffraction, dual polarisation interferometry, NMR spectroscopy, radioisotopic labeling, electron microscopy, and molecular dynamics simulations. These techniques allowed for the discovery and detailed analysis of many molecules and metabolic pathways of the cell, such as glycolysis and the Krebs cycle (citric acid cycle).

Biochemistry

Another significant historic event in biochemistry is the discovery of the gene and its role in the transfer of information in the cell. This part of biochemistry is often called molecular biology.[5] In the 1950s, James D. Watson, Francis Crick, Rosalind Franklin, and Maurice Wilkins were instrumental in solving DNA structure and suggesting its relationship with genetic transfer of information.[6] In 1958, George Beadle and Edward Tatum received the Nobel Prize for work in fungi showing that one gene produces one enzyme.[] In 1988, Colin Pitchfork was the first person convicted of murder with DNA evidence, which led to growth of forensic science.[] More recently, Andrew Z. Fire and Craig C. Mello received the 2006 Nobel Prize for discovering the role of RNA interference (RNAi), in the silencing of gene expression.[]

Starting materials: the chemical elements of life Around two dozen of the 92 naturally occurring chemical elements are essential to various kinds of biological life. Most rare elements on Earth are not needed by life (exceptions being selenium and iodine), while a few common ones (aluminum and titanium) are not used. Most organisms share element needs, but there are a few differences between plants and animals. For example ocean algae use bromine but land plants and animals seem to need none. All animals require sodium, but some plants do not. Plants need boron and silicon, but animals may not (or may need ultra-small amounts). Just six elements—carbon, hydrogen, nitrogen, oxygen, calcium, and phosphorus—make up almost 99% of the mass of a human body (see composition of the human body for a complete list). In addition to the six major elements that compose most of the human body, humans require smaller amounts of possibly 18 more.[7]

Biomolecules The four main classes of molecules in biochemistry are carbohydrates, lipids, proteins, and nucleic acids. Many biological molecules are polymers: in this terminology, monomers are relatively small micromolecules that are linked together to create large macromolecules, which are known as polymers. When monomers are linked together to synthesize a biological polymer, they undergo a process called dehydration synthesis. Different macromolecules can assemble in larger complexes, often needed for biological activity.

Carbohydrates Carbohydrates are made from monomers called monosaccharides. Some of these monosaccharides include glucose (C6H12O6), fructose (C6H12O6), and deoxyribose (C5H10O4). When two monosaccharides undergo dehydration synthesis, water is produced, as two hydrogen atoms and one oxygen atom are lost from the two monosaccharides' hydroxyl group. A molecule of sucrose (glucose + fructose), a disaccharide.

Biochemistry

Lipids

A triglyceride with a glycerol molecule on the left and three fatty acids coming off it.

Lipids are usually made from one molecule of glycerol combined with other molecules. In triglycerides, the main group of bulk lipids, there is one molecule of glycerol and three fatty acids. Fatty acids are considered the monomer in that case, and may be saturated (no double bonds in the carbon chain) or unsaturated (one or more double bonds in the carbon chain). Lipids, especially phospholipids, are also used in various pharmaceutical products, either as co-solubilisers (e.g., in parenteral infusions) or else as drug carrier components (e.g., in a liposome or transfersome).

Proteins Proteins are very large molecules – macro-biopolymers – made from monomers called amino acids. There are 20 standard amino acids, each containing a carboxyl group, an amino group, and a side-chain (known as an "R" group). The "R" group is what makes each amino acid different, and the properties of the side-chains greatly influence the overall three-dimensional conformation of a protein. When amino acids combine, they form a special bond called a peptide bond through dehydration synthesis, and become a polypeptide, or protein.

The general structure of an α-amino acid, with the amino group on the left and the carboxyl group on the right.

In order to determine whether two proteins are related, or in other words to decide whether they are homologous or not, scientists use sequence-comparison methods. Methods like Sequence Alignments and Structural Alignments are powerful tools that help scientists identify homologies between related molecules.[]

The relevance of finding homologies among proteins goes beyond forming an evolutionary pattern of protein families. By finding how similar two protein sequences are, we acquire knowledge about their structure and therefore their function.

Biochemistry

Nucleic acids Nucleic acids are the molecules that make up DNA, an extremely important substance that all cellular organisms use to store their genetic information. The most common nucleic acids are deoxyribonucleic acid and ribonucleic acid. Their monomers are called nucleotides. The most common nucleotides are adenine, cytosine, guanine, thymine, and uracil. Adenine binds with thymine and uracil; Thymine binds only with adenine; and cytosine and guanine can bind only with each other.

Carbohydrates

The structure of deoxyribonucleic acid (DNA), the picture shows the monomers being put together.

The function of carbohydrates includes energy storage and providing structure. Sugars are carbohydrates, but not all carbohydrates are sugars. There are more carbohydrates on Earth than any other known type of biomolecule; they are used to store energy and genetic information, as well as play important roles in cell to cell interactions and communications.

Monosaccharides The simplest type of carbohydrate is a monosaccharide, which among other properties contains carbon, hydrogen, and oxygen, mostly in a ratio of 1:2:1 (generalized formula CnH2nOn, where n is at least 3). Glucose, one of the most important carbohydrates, is an example of a monosaccharide. So is fructose, the sugar commonly associated with the sweet taste of fruits.[][a] Some carbohydrates (especially after condensation to oligo- and polysaccharides) contain less carbon Glucose relative to H and O, which still are present in 2:1 (H:O) ratio. Monosaccharides can be grouped into aldoses (having an aldehyde group at the end of the chain, e.g. glucose) and ketoses (having a keto group in their chain; e.g. fructose). Both aldoses and ketoses occur in an equilibrium (starting with chain lengths of C4) cyclic forms. These are generated by bond formation between one of the hydroxyl groups of the sugar chain with the carbon of the aldehyde or keto group to form a hemiacetal bond. This leads to saturated five-membered (in furanoses) or six-membered (in pyranoses) heterocyclic rings containing one O as heteroatom.

Disaccharides Two monosaccharides can be joined together using dehydration synthesis, in which a hydrogen atom is removed from the end of one molecule and a hydroxyl group (—OH) is removed from the other; the remaining residues are then attached at the sites from which the atoms were removed. The H—OH or H2O is then released as a molecule of water, hence the term dehydration. The new molecule, consisting of two monosaccharides, is called a disaccharide and is conjoined together by a glycosidic or ether bond. The reverse reaction can also

Sucrose: ordinary table sugar and probably the most familiar carbohydrate.

Biochemistry

occur, using a molecule of water to split up a disaccharide and break the glycosidic bond; this is termed hydrolysis. The most well-known disaccharide is sucrose, ordinary sugar (in scientific contexts, called table sugar or cane sugar to differentiate it from other sugars). Sucrose consists of a glucose molecule and a fructose molecule joined together. Another important disaccharide is lactose, consisting of a glucose molecule and a galactose molecule. As most humans age, the production of lactase, the enzyme that hydrolyzes lactose back into glucose and galactose, typically decreases. This results in lactase deficiency, also called lactose intolerance. Sugar polymers are characterised by having reducing or non-reducing ends. A reducing end of a carbohydrate is a carbon atom that can be in equilibrium with the open-chain aldehyde or keto form. If the joining of monomers takes place at such a carbon atom, the free hydroxy group of the pyranose or furanose form is exchanged with an OH-side-chain of another sugar, yielding a full acetal. This prevents opening of the chain to the aldehyde or keto form and renders the modified residue non-reducing. Lactose contains a reducing end at its glucose moiety, whereas the galactose moiety form a full acetal with the C4-OH group of glucose. Saccharose does not have a reducing end because of full acetal formation between the aldehyde carbon of glucose (C1) and the keto carbon of fructose (C2).

Oligosaccharides and polysaccharides When a few (around three to six) monosaccharides are joined together, it is called an oligosaccharide (oligo- meaning "few"). These molecules tend to be used as markers and signals, as well as having some other uses. Many monosaccharides joined together make a polysaccharide. They can be joined together in one long linear chain, or they may be branched. Two of the most common polysaccharides are cellulose and glycogen, both consisting of repeating glucose monomers.

Cellulose as polymer of β-D-glucose

• Cellulose is made by plants and is an important structural component of their cell walls. Humans can neither manufacture nor digest it. • Glycogen, on the other hand, is an animal carbohydrate; humans and other animals use it as a form of energy storage.

Use of carbohydrates as an energy source Glucose is the major energy source in most life forms. For instance, polysaccharides are broken down into their monomers (glycogen phosphorylase removes glucose residues from glycogen). Disaccharides like lactose or sucrose are cleaved into their two component monosaccharides. Glycolysis (anaerobic) Glucose is mainly metabolized by a very important ten-step pathway called glycolysis, the net result of which is to break down one molecule of glucose into two molecules of pyruvate; this also produces a net two molecules of ATP, the energy currency of cells, along with two reducing equivalents in the form of converting NAD+ to NADH. This does not require oxygen; if no oxygen is available (or the cell cannot use oxygen), the NAD is restored by converting the pyruvate to lactate (lactic acid) (e.g., in humans) or to ethanol plus carbon dioxide (e.g., in yeast). Other monosaccharides like galactose and fructose can be converted into intermediates of the glycolytic pathway.[8]

Biochemistry

Aerobic In aerobic cells with sufficient oxygen, as in most human cells, the pyruvate is further metabolized. It is irreversibly converted to acetyl-CoA, giving off one carbon atom as the waste product carbon dioxide, generating another reducing equivalent as NADH. The two molecules acetyl-CoA (from one molecule of glucose) then enter the citric acid cycle, producing two more molecules of ATP, six more NADH molecules and two reduced (ubi)quinones (via FADH2 as enzyme-bound cofactor), and releasing the remaining carbon atoms as carbon dioxide. The produced NADH and quinol molecules then feed into the enzyme complexes of the respiratory chain, an electron transport system transferring the electrons ultimately to oxygen and conserving the released energy in the form of a proton gradient over a membrane (inner mitochondrial membrane in eukaryotes). Thus, oxygen is reduced to water and the original electron acceptors NAD+ and quinone are regenerated. This is why humans breathe in oxygen and breathe out carbon dioxide. The energy released from transferring the electrons from high-energy states in NADH and quinol is conserved first as proton gradient and converted to ATP via ATP synthase. This generates an additional 28 molecules of ATP (24 from the 8 NADH + 4 from the 2 quinols), totaling to 32 molecules of ATP conserved per degraded glucose (two from glycolysis + two from the citrate cycle). It is clear that using oxygen to completely oxidize glucose provides an organism with far more energy than any oxygen-independent metabolic feature, and this is thought to be the reason why complex life appeared only after Earth's atmosphere accumulated large amounts of oxygen. Gluconeogenesis In vertebrates, vigorously contracting skeletal muscles (during weightlifting or sprinting, for example) do not receive enough oxygen to meet the energy demand, and so they shift to anaerobic metabolism, converting glucose to lactate. The liver regenerates the glucose, using a process called gluconeogenesis. This process is not quite the opposite of glycolysis, and actually requires three times the amount of energy gained from glycolysis (six molecules of ATP are used, compared to the two gained in glycolysis). Analogous to the above reactions, the glucose produced can then undergo glycolysis in tissues that need energy, be stored as glycogen (or starch in plants), or be converted to other monosaccharides or joined into di- or oligosaccharides. The combined pathways of glycolysis during exercise, lactate's crossing via the bloodstream to the liver, subsequent gluconeogenesis and release of glucose into the bloodstream is called the Cori cycle.[9]

Proteins Like carbohydrates, some proteins perform largely structural roles. For instance, movements of the proteins actin and myosin ultimately are responsible for the contraction of skeletal muscle. One property many proteins have is that they specifically bind to a certain molecule or class of molecules—they may be extremely selective in what they bind. Antibodies are an example of proteins that attach to one specific type of molecule. In fact, the enzyme-linked immunosorbent assay (ELISA), which uses antibodies, is currently one of the most sensitive tests modern medicine uses to detect various biomolecules. Probably the most important proteins, however, are the enzymes. These molecules recognize specific reactant molecules called substrates; they then catalyze the reaction between them. By lowering the activation energy, the enzyme speeds up that reaction by a rate of 1011 or more: a reaction that would normally take over 3,000 years to complete spontaneously might take less than a second with an enzyme.

A schematic of hemoglobin. The red and blue ribbons represent the protein globin; the green structures are the heme groups.

The enzyme itself is not used up in the process, and is free to catalyze the same reaction with a new set of substrates. Using various modifiers, the activity of the enzyme can be regulated, enabling control of the biochemistry of the cell

Biochemistry as a whole. In essence, proteins are chains of amino acids. An amino acid consists of a carbon atom bound to four groups. One is an amino group, —NH2, and one is a carboxylic acid group, —COOH (although these exist as —NH3+ and —COO− under physiologic conditions). The third is a simple hydrogen atom. The fourth is commonly denoted "—R" and is different for each amino acid. There are twenty standard amino acids. Some of these have functions by themselves or in a modified form; for instance, glutamate functions as an important neurotransmitter. Amino acids can be joined together via a peptide bond. In this dehydration synthesis, a water molecule is removed and the peptide bond connects the nitrogen of one amino acid's amino group to the carbon of the other's Generic amino acids (1) in neutral form, (2) as they exist physiologically, and (3) joined carboxylic acid group. The resulting together as a dipeptide. molecule is called a dipeptide, and short stretches of amino acids (usually, fewer than thirty) are called peptides or polypeptides. Longer stretches merit the title proteins. As an example, the important blood serum protein albumin contains 585 amino acid residues.[] The structure of proteins is traditionally described in a hierarchy of four levels. The primary structure of a protein simply consists of its linear sequence of amino acids; for instance, "alanine-glycine-tryptophan-serine-glutamate-asparagine-glycine-lysine-…". Secondary structure is concerned with local morphology (morphology being the study of structure). Some combinations of amino acids will tend to curl up in a coil called an α-helix or into a sheet called a β-sheet; some α-helixes can be seen in the hemoglobin schematic above. Tertiary structure is the entire three-dimensional shape of the protein. This shape is determined by the sequence of amino acids. In fact, a single change can change the entire structure. The alpha chain of hemoglobin contains 146 amino acid residues; substitution of the glutamate residue at position 6 with a valine residue changes the behavior of hemoglobin so much that it results in sickle-cell disease. Finally, quaternary structure is concerned with the structure of a protein with multiple peptide subunits, like hemoglobin with its four subunits. Not all proteins have more than one subunit.[10] Ingested proteins are usually broken up into single amino acids or dipeptides in the small intestine, and then absorbed. They can then be joined together to make new proteins. Intermediate products of glycolysis, the citric acid cycle, and the pentose phosphate pathway can be used to make all twenty amino acids, and most bacteria and plants possess all the necessary enzymes to synthesize them. Humans and other mammals, however, can synthesize only half of them. They cannot synthesize isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. These are the essential amino acids, since it is essential to ingest them. Mammals do possess the enzymes to synthesize alanine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine, the nonessential amino acids. While they can synthesize arginine and histidine, they cannot produce it in sufficient amounts for young, growing animals, and so these are often considered essential amino acids. If the amino group is removed from an amino acid, it leaves behind a carbon skeleton called an α-keto acid. Enzymes called transaminases can easily transfer the amino group from one amino acid (making it an α-keto acid) to another α-keto acid (making it an amino acid). This is important in the biosynthesis of amino acids, as for many of the pathways, intermediates from other biochemical pathways are converted to the α-keto acid skeleton, and then an amino group is added, often via transamination. The amino acids may then be linked together to make a protein.[11] A similar process is used to break down proteins. It is first hydrolyzed into its component amino acids. Free ammonia (NH3), existing as the ammonium ion (NH4+) in blood, is toxic to life forms. A suitable method for excreting it must therefore exist. Different strategies have evolved in different animals, depending on the animals' needs. Unicellular organisms, of course, simply release the ammonia into the environment. Likewise, bony fish can

Biochemistry release the ammonia into the water where it is quickly diluted. In general, mammals convert the ammonia into urea, via the urea cycle.[]

Lipids The term lipid comprises a diverse range of molecules and to some extent is a catchall for relatively water-insoluble or nonpolar compounds of biological origin, including waxes, fatty acids, fatty-acid derived phospholipids, sphingolipids, glycolipids, and terpenoids (e.g., retinoids and steroids). Some lipids are linear aliphatic molecules, while others have ring structures. Some are aromatic, while others are not. Some are flexible, while others are rigid.[12] Most lipids have some polar character in addition to being largely nonpolar. In general, the bulk of their structure is nonpolar or hydrophobic ("water-fearing"), meaning that it does not interact well with polar solvents like water. Another part of their structure is polar or hydrophilic ("water-loving") and will tend to associate with polar solvents like water. This makes them amphiphilic molecules (having both hydrophobic and hydrophilic portions). In the case of cholesterol, the polar group is a mere -OH (hydroxyl or alcohol). In the case of phospholipids, the polar groups are considerably larger and more polar, as described below. Lipids are an integral part of our daily diet. Most oils and milk products that we use for cooking and eating like butter, cheese, ghee etc., are composed of fats. Vegetable oils are rich in various polyunsaturated fatty acids (PUFA). Lipid-containing foods undergo digestion within the body and are broken into fatty acids and glycerol, which are the final degradation products of fats and lipids.

Nucleic acids A nucleic acid is a complex, high-molecular-weight biochemical macromolecule composed of nucleotide chains that convey genetic information. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are found in all living cells and viruses. Aside from the genetic material of the cell, nucleic acids often play a role as second messengers, as well as forming the base molecule for adenosine triphosphate, the primary energy-carrier molecule found in all living organisms. Nucleic acid, so called because of its prevalence in cellular nuclei, is the generic name of the family of biopolymers. The monomers are called nucleotides, and each consists of three components: a nitrogenous heterocyclic base (either a purine or a pyrimidine), a pentose sugar, and a phosphate group. Different nucleic acid types differ in the specific sugar found in their chain (e.g., DNA or deoxyribonucleic acid contains 2-deoxyriboses). Also, the nitrogenous bases possible in the two nucleic acids are different: adenine, cytosine, and guanine occur in both RNA and DNA, while thymine occurs only in DNA and uracil occurs in RNA.[13]

Biochemistry

Relationship to other "molecular-scale" biological sciences Researchers in biochemistry use specific techniques native to biochemistry, but increasingly combine these with techniques and ideas developed in the fields of genetics, molecular biology and biophysics. There has never been a hard-line between these disciplines in terms of content and technique. Today, the terms molecular biology and biochemistry are nearly interchangeable. The following figure is a schematic that depicts one possible view of the relationship between the fields: • Biochemistry is the study of the chemical substances and vital processes occurring in living organisms. Biochemists focus heavily on the role, function, and structure of biomolecules. The study of the chemistry behind biological processes and the synthesis of biologically active molecules are examples of biochemistry.

Schematic relationship between biochemistry, genetics, and molecular biology

• Genetics is the study of the effect of genetic differences on organisms. Often this can be inferred by the absence of a normal component (e.g., one gene). The study of "mutants" – organisms with a changed gene that leads to the organism being different with respect to the so-called "wild type" or normal phenotype. Genetic interactions (epistasis) can often confound simple interpretations of such "knock-out" or "knock-in" studies. • Molecular biology is the study of molecular underpinnings of the process of replication, transcription and translation of the genetic material. The central dogma of molecular biology where genetic material is transcribed into RNA and then translated into protein, despite being an oversimplified picture of molecular biology, still provides a good starting point for understanding the field. This picture, however, is undergoing revision in light of emerging novel roles for RNA.[] • Chemical Biology seeks to develop new tools based on small molecules that allow minimal perturbation of biological systems while providing detailed information about their function. Further, chemical biology employs biological systems to create non-natural hybrids between biomolecules and synthetic devices (for example emptied viral capsids that can deliver gene therapy or drug molecules).

Notes a. ^ Fructose is not the only sugar found in fruits. Glucose and sucrose are also found in varying quantities in various fruits, and indeed sometimes exceed the fructose present. For example, 32% of the edible portion of date is glucose, compared with 23.70% fructose and 8.20% sucrose. However, peaches contain more sucrose (6.66%) than they do fructose (0.93%) or glucose (1.47%).[14]

References [1] http:/ / portal. acs. org/ portal/ acs/ corg/ content?_nfpb=true& _pageLabel=PP_ARTICLEMAIN& node_id=1188& content_id=CTP_003379& use_sec=true& sec_url_var=region1& __uuid=aa3f2aa3-8047-4fa2-88b8-32ffcad3a93e [2] http:/ / www. biochemistry. org/ Education/ Careers/ Schoolsandcolleges/ Whatisbiochemistry. aspx [3] Hunter (2000), p. 75. [4] Hunter (2000), pp. 96–98. [5] Tropp (2012), p. 2. [6] Tropp (2012), pp. 19–20.

Biochemistry [7] Ultratrace minerals. Authors: Nielsen, Forrest H. USDA, ARS Source: Modern nutrition in health and disease / editors, Maurice E. Shils ... et al.. Baltimore : Williams & Wilkins, c1999., p. 283-303. Issue Date: 1999 URI: (http:/ / hdl. handle. net/ 10113/ 46493) [8] Fromm and Hargrove (2012), pp. 163–180. [9] Fromm and Hargrove (2012), pp. 183–194. [10] Fromm and Hargrove (2012), pp. 35–51. [11] Fromm and Hargrove (2012), pp. 279–292. [12] Fromm and Hargrove (2012), pp. 22–27. [13] Tropp (2012), pp. 5–9. [14] Whiting, G.C. (1970), p.5

Cited literature • Fromm, Herbert J.; Hargrove, Mark (2012). Essentials of Biochemistry. Springer. ISBN978-3-642-19623-2. • Hunter, Graeme K. (2000). Vital Forces: The Discovery of the Molecular Basis of Life. Academic Press. ISBN978-0-12-361811-5. • Tropp, Burton E. (2012). Molecular Biology (4th ed.). Jones & Bartlett Learning. ISBN978-1-4496-0091-4.

External links • The Virtual Library of Biochemistry and Cell Biology (http://www.biochemweb.org/) • Biochemistry, 5th ed. (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowTOC&rid=stryer. TOC&depth=2) Full text of Berg, Tymoczko, and Stryer, courtesy of NCBI. • Biochemistry, 2nd ed. (http://www.web.virginia.edu/Heidi/home.htm) Full text of Garrett and Grisham. • Biochemistry Animation (http://www.1lec.com/Biochemistry/) (Narrated Flash animations.) • SystemsX.ch - The Swiss Initiative in Systems Biology (http://www.systemsX.ch/) • Biochemistry Online Resources (http://www.icademic.org/97445/Biochemistry/) – Lists of Biochemistry departments, websites, journals, books and reviews, employment opportunities and events. • biochemical families: carbohydrates • alcohols • glycoproteins • glycosides • lipids • • • • •

eicosanoids fatty acids / intermediates phospholipids sphingolipids steroids

• nucleic acids • constituents / intermediates • proteins • amino acids / intermediates • tetrapyrroles / intermediates

Cells

Cells The cell is the basic structural, functional and biological unit of all known living organisms. It is the smallest unit of life that is classified as a living thing (except virus, which consists only from DNA/RNA covered by protein and lipids), and is often called the building block of life. It consists of a protoplasm enclosed within a membrane, which contains many biomolecules such as proteins and nucleic acids. [1] Organisms can be classified as unicellular (consisting of a single cell; including most bacteria) or multicellular (including plants and animals).

Allium cells in different phases of the cell cycle

While the number of cells in plants and animals varies from species to species, Humans contain about 100 trillion (1014) cells.[2] Most plant and animal cells are between 1 and 100micrometres and therefore are visible only under the microscope.[3] The cell was discovered by Robert Hooke in 1665. The cell theory, first The cells of eukaryotes (left) and prokaryotes (right) developed in 1839 by Matthias Jakob Schleiden and Theodor Schwann, states that all organisms are composed of one or more cells, that all cells come from preexisting cells, that vital functions of an organism occur within cells, and that all cells contain the hereditary information necessary for regulating cell functions and for transmitting information to the next generation of cells.[4] Cells emerged on planet Earth at least 4.0–4.3 billion years ago. The word cell comes from the Latin cella, meaning "small room".[5] The descriptive term for the smallest living biological structure was coined by Robert Hooke in a book he published in 1665 when he compared the cork cells he saw through his microscope to the small rooms monks lived in.[6]

Cells

Anatomy There are two types of cells: Eukaryote and prokaryote. Prokaryotic cells are usually independent, while eukaryotic cells can either exist as a single celled organism or be found in multicellular organisms.

Table 1: Comparison of features of prokaryotic and eukaryotic cells Prokaryotes Typical organisms Typical size Type of nucleus DNA

bacteria, archaea []

protists, fungi, plants, animals [] ~ 10–100 µm (sperm cells, apart from the tail, are smaller)

~ 1–5 µm

nucleoid region; no real nucleus real nucleus with double membrane circular (usually)

RNA-/protein-synthesis coupled in cytoplasm

Ribosomes

Eukaryotes

50S+30S

linear molecules (chromosomes) with histone proteins RNA-synthesis inside the nucleus protein synthesis in cytoplasm 60S+40S

Cytoplasmatic structure very few structures

highly structured by endomembranes and a cytoskeleton

Cell movement

flagella made of flagellin

flagella and cilia containing microtubules; lamellipodia and filopodia containing actin

Mitochondria

none

one to several thousand (though some lack mitochondria)

Chloroplasts

none

in algae and plants

Organization

usually single cells

single cells, colonies, higher multicellular organisms with specialized cells

Cell division

Binary fission (simple division) Mitosis (fission or budding) Meiosis

Prokaryotic cells The prokaryote cell is simpler, and therefore smaller, than a eukaryote cell, lacking a nucleus and most of the other organelles of eukaryotes. There are two kinds of prokaryotes: bacteria and archaea; these share a similar structure. The nuclear material of a prokaryotic cell consists of a single chromosome that is in direct contact with the cytoplasm. Here, the undefined nuclear region in the cytoplasm is called the nucleoid. A prokaryotic cell architectural regions:

has

three

• On the outside, flagella and pili Diagram of a typical prokaryotic cell project from the cell's surface. These are structures (not present in all prokaryotes) made of proteins that facilitate movement and communication between cells.

Cells

• Enclosing the cell is the cell envelope – generally consisting of a cell wall covering a plasma membrane though some bacteria also have a further covering layer called a capsule. The envelope gives rigidity to the cell and separates the interior of the cell from its environment, serving as a protective filter. Though most prokaryotes have a cell wall, there are exceptions such as Mycoplasma (bacteria) and Thermoplasma (archaea). The cell wall consists of peptidoglycan in bacteria, and acts as an additional barrier against exterior forces. It also prevents the cell from expanding and finally bursting (cytolysis) from osmotic pressure against a hypotonic environment. Some eukaryote cells (plant cells and fungal cells) also have a cell wall. • Inside the cell is the cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions. A prokaryotic chromosome is usually a circular molecule (an exception is that of the bacterium Borrelia burgdorferi, which causes Lyme disease).[7] Though not forming a nucleus, the DNA is condensed in a nucleoid. Prokaryotes can carry extrachromosomal DNA elements called plasmids, which are usually circular. Plasmids enable additional functions, such as antibiotic resistance.

Eukaryotic cells Plants, animals, fungi, slime moulds, protozoa, and algae are all eukaryotic. These cells are about 15 times wider than a typical prokaryote and can be as much as 1000 times greater in volume. The major difference between prokaryotes and eukaryotes is that eukaryotic cells contain membrane-bound compartments in which specific metabolic activities take place. Most important among these is a cell nucleus, a membrane-delineated compartment that houses the eukaryotic cell's DNA. This nucleus gives the eukaryote its name, which means "true nucleus." Other differences include: • The plasma membrane resembles that of prokaryotes in function, with minor differences in the setup. Cell walls may or may not be present. • The eukaryotic DNA is organized in one or more linear molecules, called chromosomes, which are associated with histone proteins. All chromosomal DNA is stored in the cell nucleus, separated from the cytoplasm by a membrane. Some eukaryotic organelles such as mitochondria also contain some DNA. • Many eukaryotic cells are ciliated with primary cilia. Primary cilia play important roles in chemosensation, mechanosensation, and thermosensation. Cilia may thus be "viewed as sensory cellular antennae that coordinate a large number of cellular signaling pathways, sometimes coupling the signaling to ciliary motility or alternatively to cell division and differentiation."[] • Eukaryotes can move using motile cilia or flagella. The flagella are more complex than those of prokaryotes.

Structure of a typical animal cell

Structure of a typical plant cell

Cells

Table 2: Comparison of structures between animal and plant cells Typical animal cell Organelles • • • • • • • • • • •

Typical plant cell

Nucleus

•

Nucleus

• Nucleolus (within nucleus) Rough endoplasmic reticulum (ER) Smooth ER Ribosomes Cytoskeleton Golgi apparatus Cytoplasm Mitochondria Vesicles Lysosomes Centrosome

• • • • • • • • • •

• Nucleolus (within nucleus) Rough ER Smooth ER Ribosomes Cytoskeleton Golgi apparatus (dictiosomes) Cytoplasm Mitochondria Plastids and its derivatives Vacuole(s) Cell wall

•

Centrioles

Subcellular components All cells, whether prokaryotic or eukaryotic, have a membrane that envelops the cell, separates its interior from its environment, regulates what moves in and out (selectively permeable), and maintains the electric potential of the cell. Inside the membrane, a salty cytoplasm takes up most of the cell volume. All cells (except red blood cells which lack a cell nucleus and most organelles to accommodate maximum space for hemoglobin) possess DNA, the hereditary material of genes, and RNA, containing the information necessary to build various proteins such as enzymes, the cell's primary machinery. There are also other kinds of biomolecules in cells. This article lists these primary components of the cell, then briefly describe their function.

Membrane The cytoplasm of a cell is surrounded by a cell membrane or plasma membrane. The plasma membrane in plants and prokaryotes is usually covered by a cell wall. This membrane serves to separate and protect a cell from its surrounding environment and is made mostly from a double layer of lipids (hydrophobic fat-like molecules) and hydrophilic phosphorus molecules. Hence, the layer is called a phospholipid bilayer, or sometimes a fluid mosaic membrane. Embedded within this membrane is a variety of protein molecules that act as channels and pumps that move different molecules into and out of the cell. The membrane is said to be 'semi-permeable', in that it can either let a substance (molecule or ion) pass through freely, pass through to a limited extent or not pass through at all. Cell surface membranes also contain receptor proteins that allow cells to detect external signaling molecules such as hormones.

Cells

Cytoskeleton The cytoskeleton acts to organize and maintain the cell's shape; anchors organelles in place; helps during endocytosis, the uptake of external materials by a cell, and cytokinesis, the separation of daughter cells after cell division; and moves parts of the cell in processes of growth and mobility. The eukaryotic cytoskeleton is composed of microfilaments, intermediate filaments and microtubules. There are a great number of proteins associated with them, each controlling a cell's structure by directing, bundling, and aligning filaments. The prokaryotic cytoskeleton is less well-studied but is involved in the maintenance of cell shape, polarity and cytokinesis.[8]

Genetic material

Bovine Pulmonary Artery Endothelial cell: nuclei stained blue, mitochondria stained red, and F-actin, an important component in microfilaments, stained green. Cell imaged on a fluorescent microscope.

Two different kinds of genetic material exist: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Most cells use DNA for their long-term information storage. The biological information contained in an organism is encoded in its DNA sequence. RNA is used for information transport (e.g., mRNA) and enzymatic functions (e.g., ribosomal RNA). Transfer RNA (tRNA) molecules are used to add amino acids during protein translation. Prokaryotic genetic material is organized in a simple circular DNA molecule (the bacterial chromosome) in the nucleoid region of the cytoplasm. Eukaryotic genetic material is divided into different, linear molecules called chromosomes inside a discrete nucleus, usually with additional genetic material in some organelles like mitochondria and chloroplasts (see endosymbiotic theory). A human cell has genetic material contained in the cell nucleus (the nuclear genome) and in the mitochondria (the mitochondrial genome). In humans the nuclear genome is divided into 46 linear DNA molecules called chromosomes, including 22 homologous chromosome pairs and a pair of sex chromosomes. The mitochondrial genome is a circular DNA molecule distinct from the nuclear DNA. Although the mitochondrial DNA is very small compared to nuclear chromosomes, it codes for 13 proteins involved in mitochondrial energy production and specific tRNAs. Foreign genetic material (most commonly DNA) can also be artificially introduced into the cell by a process called transfection. This can be transient, if the DNA is not inserted into the cell's genome, or stable, if it is. Certain viruses also insert their genetic material into the genome.

Organelles The human body contains many different organs, such as the heart, lung, and kidney, with each organ performing a different function. Cells also have a set of "little organs," called organelles, that are adapted and/or specialized for carrying out one or more vital functions. Both eukaryotic and prokaryotic cells have organelles but organelles in eukaryotes are generally more complex and may be membrane bound. There are several types of organelles in a cell. Some (such as the nucleus and golgi apparatus) are typically solitary, while others (such as mitochondria, peroxisomes and lysosomes) can be numerous (hundreds to thousands). The cytosol is the gelatinous fluid that fills the cell and surrounds the organelles.

Cells

• Cell nucleus – eukaryotes only - A cell's information center, the cell nucleus is the most conspicuous organelle found in a eukaryotic cell. It houses the cell's chromosomes, and is the place where almost all DNA replication and RNA synthesis (transcription) occur. The nucleus is spherical and separated from the cytoplasm by a double membrane called the nuclear envelope. The nuclear envelope isolates and protects a cell's DNA from various molecules that could accidentally damage its structure or interfere with its processing. During processing, DNA is transcribed, or copied into a special RNA, called messenger RNA (mRNA). This mRNA is then transported out of the nucleus, where it is translated into a specific protein molecule. The nucleolus is a specialized region within the nucleus where ribosome subunits are assembled. In prokaryotes, DNA processing takes place in the cytoplasm.

Diagram of a cell nucleus

• Mitochondria and Chloroplasts – eukaryotes only - the power generators: Mitochondria are self-replicating organelles that occur in various numbers, shapes, and sizes in the cytoplasm of all eukaryotic cells. Mitochondria play a critical role in generating energy in the eukaryotic cell. Mitochondria generate the cell's energy by oxidative phosphorylation, using oxygen to release energy stored in cellular nutrients (typically pertaining to glucose) to generate ATP. Mitochondria multiply by splitting in two. Respiration occurs in the cell mitochondria. Chloroplasts can only be found in plants and algae, and they capture the sun's energy to make ATP. • Endoplasmic reticulum – eukaryotes only: The endoplasmic reticulum (ER) is the transport network for molecules targeted for certain modifications and specific destinations, as compared to molecules that float freely in the cytoplasm. The ER has two forms: the rough ER, which has ribosomes on its surface and secretes proteins into the cytoplasm, and the smooth ER, which lacks them. Smooth ER plays a role in calcium sequestration and release.[citation needed]

• Golgi apparatus – eukaryotes only : The primary function of the Golgi apparatus is to process and package the macromolecules such as proteins and lipids that are synthesized by the cell.[citation needed] • Ribosomes: The ribosome is a large complex of RNA and protein Diagram of an endomembrane system molecules. They each consist of two subunits, and act as an assembly line where RNA from the nucleus is used to synthesise proteins from amino acids. Ribosomes can be found either floating freely or bound to a membrane (the rough endoplasmatic reticulum in eukaryotes, or the cell membrane in prokaryotes).[9] • Lysosomes and Peroxisomes – eukaryotes only: Lysosomes contain digestive enzymes (acid hydrolases). They digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria. Peroxisomes have enzymes that rid the cell of toxic peroxides. The cell could not house these destructive enzymes if they were not contained in a membrane-bound system.[citation needed] • Centrosome – the cytoskeleton organiser: The centrosome produces the microtubules of a cell – a key component of the cytoskeleton. It directs the transport through the ER and the Golgi apparatus. Centrosomes are composed of two centrioles, which separate during cell division and help in the formation of the mitotic spindle. A single centrosome is present in the animal cells. They are also found in some fungi and algae cells.[citation needed]

Cells • Vacuoles: Vacuoles store food and waste. Some vacuoles store extra water. They are often described as liquid filled space and are surrounded by a membrane. Some cells, most notably Amoeba, have contractile vacuoles, which can pump water out of the cell if there is too much water. The vacuoles of eukaryotic cells are usually larger in those of plants than animals.[citation needed]

Structures outside the cell membrane Many cells also have structures which exist wholly or partially outside the cell membrane. These structures are notable because they are not protected from the external environment by the impermeable cell membrane. In order to assemble these structures export processes to carry macromolecules across the cell membrane must be used.

Cell wall Many types of prokaryotic and eukaryotic cell have a cell wall. The cell wall acts to protect the cell mechanically and chemically from its environment, and is an additional layer of protection to the cell membrane. Different types of cell have cell walls made up of different materials; plant cell walls are primarily made up of pectin, fungi cell walls are made up of chitin and bacteria cell walls are made up of peptidoglycan.

Prokaryotic Capsule A gelatinous capsule is present in some bacteria outside the cell membrane and cell wall. The capsule may be polysaccharide as in pneumococci, meningococci or polypeptide as Bacillus anthracis or hyaluronic acid as in streptococci.[citation needed] Capsules are not marked by normal staining protocols and can be detected by special stain.[citation needed] Flagella Flagella are organelles for cellular mobility. The bacterial flagellum stretches from cytoplasm through the cell membrane(s) and extrudes through the cell wall. They are long and thick thread-like appendages, protein in nature. Are most commonly found in bacteria cells but are found in animal cells as well. Fimbriae (pili) They are short and thin hair like filaments, formed of protein called pilin (antigenic). Fimbriae are responsible for attachment of bacteria to specific receptors of human cell (adherence). There are special types of pili called (sex pili) involved in conjunction.[citation needed]

Growth and metabolism Between successive cell divisions, cells grow through the functioning of cellular metabolism. Cell metabolism is the process by which individual cells process nutrient molecules. Metabolism has two distinct divisions: catabolism, in which the cell breaks down complex molecules to produce energy and reducing power, and anabolism, in which the cell uses energy and reducing power to construct complex molecules and perform other biological functions. Complex sugars consumed by the organism can be broken down into a less chemically complex sugar molecule called glucose. Once inside the cell, glucose is broken down to make adenosine triphosphate (ATP), a form of energy, through two different pathways. The first pathway, glycolysis, requires no oxygen and is referred to as anaerobic metabolism. Each reaction is designed to produce some hydrogen ions that can then be used to make energy packets (ATP). In prokaryotes, glycolysis is the only method used for converting energy.

Cells

The second pathway, called the Krebs cycle, or citric acid cycle, occurs inside the mitochondria and can generate enough ATP to run all the cell functions.[citation needed]

Self-replication Cell division involves a single cell (called a mother cell) dividing into two daughter cells. This leads to growth in multicellular organisms (the growth of tissue) and to procreation (vegetative reproduction) in unicellular organisms. Prokaryotic cells divide by binary fission. Eukaryotic cells usually undergo a process of nuclear division, called mitosis, followed by division of the cell, called cytokinesis. A diploid cell may also undergo meiosis to produce haploid cells, usually four. Haploid cells serve as gametes in multicellular organisms, fusing to form new diploid cells. DNA replication, or the process of duplicating a cell's genome, always happens when a cell divides through mitosis or binary fission. In meiosis, the DNA is replicated only once, while the cell divides twice. DNA replication only occurs before meiosis I. DNA replication does not occur when the cells divide the second time, in meiosis II.[10] Replication, like all cellular activities, requires specialized proteins for carrying out the job.

Protein synthesis Cells are capable of synthesizing new proteins, which are essential for the modulation and maintenance of cellular activities. This process involves the formation of new protein molecules from amino acid building blocks based on information encoded in DNA/RNA. Protein synthesis generally consists of two major steps: transcription and translation. Transcription is the process where genetic information in DNA is used to produce a complementary RNA strand. This RNA strand is then processed to give messenger RNA (mRNA), which is free to migrate through the cell. mRNA molecules bind to protein-RNA complexes called ribosomes located in the cytosol, where they are translated into polypeptide sequences. The ribosome mediates the formation of a polypeptide sequence based on the mRNA sequence. The mRNA sequence directly relates to the polypeptide sequence by binding to transfer RNA (tRNA) adapter molecules in binding pockets within the ribosome. The new polypeptide then folds into a functional three-dimensional protein molecule.

An overview of protein synthesis.Within the nucleus of the cell (light blue), genes (DNA, dark blue) are transcribed into RNA. This RNA is then subject to post-transcriptional modification and control, resulting in a mature mRNA (red) that is then transported out of the nucleus and into the cytoplasm (peach), where it undergoes translation into a protein. mRNA is translated by ribosomes (purple) that match the three-base codons of the mRNA to the three-base anti-codons of the appropriate tRNA. Newly synthesized proteins (black) are often further modified, such as by binding to an effector molecule (orange), to become fully active.

Movement or motility Cells can move during many processes: such as wound healing, the immune response and cancer metastasis. For wound healing to occur, white blood cells and cells that ingest bacteria move to the wound site to kill the microorganisms that cause infection. At the same time fibroblasts (connective tissue cells) move there to remodel damaged structures. In the case of tumor development, cells from a primary tumor move away and spread to other parts of the body. Cell motility involves many receptors, crosslinking, bundling, binding, adhesion, motor and other proteins.[11] The process is divided into three steps – protrusion of the leading edge of the cell, adhesion of the leading edge and de-adhesion at the cell body

Cells and rear, and cytoskeletal contraction to pull the cell forward. Each step is driven by physical forces generated by unique segments of the cytoskeleton.[12][13]

Origins The origin of cells has to do with the origin of life, which began the history of life on Earth.

Origin of the first cell There are several theories about the origin of small molecules that could lead to life in an early Earth. One is that they came from meteorites (see Murchison meteorite). Another is that they were created at deep-sea vents. A third is that they were synthesized by lightning in a reducing atmosphere (see Miller–Urey experiment); although it is not clear if Earth had such an atmosphere. There are essentially no experimental data defining what the first self-replicating forms were. RNA is generally assumed the earliest self-replicating molecule, as it is capable of both storing genetic information and catalyzing chemical reactions (see RNA world hypothesis). But some other entity with the potential to self-replicate could have preceded RNA, like clay or peptide nucleic acid.[] Cells emerged at least 4.0–4.3 billion years ago. The current belief is that these cells were heterotrophs. An important characteristic of cells is the cell membrane, composed of a bilayer of lipids. The early cell membranes were probably more simple and permeable than modern ones, with only a single fatty acid chain per lipid. Lipids are known to spontaneously form bilayered vesicles in water, and could have preceded RNA, but the first cell membranes could also have been produced by catalytic RNA, or even have required structural proteins before they could form.[14]

Origin of eukaryotic cells The eukaryotic cell seems to have evolved from a symbiotic community of prokaryotic cells. DNA-bearing organelles like the mitochondria and the chloroplasts are almost certainly what remains of ancient symbiotic oxygen-breathing proteobacteria and cyanobacteria, respectively, where the rest of the cell appears derived from an ancestral archaean prokaryote cell—an idea called the endosymbiotic theory. There is still considerable debate about whether organelles like the hydrogenosome predated the origin of mitochondria, or viceversa: see the hydrogen hypothesis for the origin of eukaryotic cells. Sex, as the stereotyped choreography of meiosis and syngamy that persists in nearly all extant eukaryotes, may have played a role in the transition from prokaryotes to eukaryotes. An 'origin of sex as vaccination' theory suggests that the eukaryote genome accreted from prokaryan parasite genomes in numerous rounds of lateral gene transfer. Sex-as-syngamy (fusion sex) arose when infected hosts began swapping nuclearized genomes containing co-evolved, vertically transmitted symbionts that conveyed protection against horizontal infection by more virulent symbionts.[]

History of research • 1632–1723: Antonie van Leeuwenhoek teaches himself to make lenses, constructs simple microscopes and draws protozoa, such as Vorticella from rain water, and bacteria from his own mouth. • 1665: Robert Hooke discovers cells in cork, then in living plant tissue using an early compound microscope.[6] • 1839: Theodor Schwann and Matthias Jakob Schleiden elucidate the principle that plants and animals are made of cells, concluding that cells are a common unit of structure and development, and thus founding the cell theory. • 1855: Rudolf Virchow states that cells always emerge from cell divisions (omnis cellula ex cellula). • 1859: The belief that life forms can occur spontaneously (generatio spontanea) is contradicted by Louis Pasteur (1822–1895) (although Francesco Redi had performed an experiment in 1668 that suggested the same conclusion).

Cells

• 1931: Ernst Ruska builds first transmission electron microscope (TEM) at the University of Berlin. By 1935, he has built an EM with twice the resolution of a light microscope, revealing previously unresolvable organelles. • 1953: Watson and Crick made their first announcement on the double-helix structure for DNA on February 28. • 1981: Lynn Margulis published Symbiosis in Cell Evolution detailing the endosymbiotic theory.

References [1] Cell Movements and the Shaping of the Vertebrate Body (http:/ / www. ncbi. nlm. nih. gov/ entrez/ query. fcgi?cmd=Search& db=books& doptcmdl=GenBookHL& term=Cell+ Movements+ and+ the+ Shaping+ of+ the+ Vertebrate+ Body+ AND+ mboc4[book]+ AND+ 374635[uid]& rid=mboc4. section. 3919) in Chapter 21 of Molecular Biology of the Cell (http:/ / www. ncbi. nlm. nih. gov/ entrez/ query. fcgi?cmd=Search& db=books& doptcmdl=GenBookHL& term=cell+ biology+ AND+ mboc4[book]+ AND+ 373693[uid]& rid=mboc4) fourth edition, edited by Bruce Alberts (2002) published by Garland Science. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos. It is also common to describe small molecules such as amino acids as " molecular building blocks (http:/ / www. ncbi. nlm. nih. gov/ entrez/ query. fcgi?cmd=Search& db=books& doptcmdl=GenBookHL& term="all+ cells"+ AND+ mboc4[book]+ AND+ 372023[uid]& rid=mboc4. section. 4#23)". [6] "... I could exceedingly plainly perceive it to be all perforated and porous, much like a Honey-comb, but that the pores of it were not regular [..] these pores, or cells, [..] were indeed the first microscopical pores I ever saw, and perhaps, that were ever seen, for I had not met with any Writer or Person, that had made any mention of them before this. . ." – Hooke describing his observations on a thin slice of cork. Robert Hooke (http:/ / www. ucmp. berkeley. edu/ history/ hooke. html) [7] European Bioinformatics Institute, Karyn's Genomes: Borrelia burgdorferi (http:/ / www. ebi. ac. uk/ 2can/ genomes/ bacteria/ Borrelia_burgdorferi. html), part of 2can on the EBI-EMBL database. Retrieved 5 August 2012 [12] Alberts B, Johnson A, Lewis J. et al. Molecular Biology of the Cell, 4e. Garland Science. 2002 [13] Ananthakrishnan R, Ehrlicher A. The Forces Behind Cell Movement. Int J Biol Sci 2007; 3:303–317. http:/ / www. biolsci. org/ v03p0303. htm

•

This article incorporatespublic domain material from the NCBI document "Science Primer" (http://www. ncbi.nlm.nih.gov/About/primer/index.html).

External links • Inside the Cell (http://publications.nigms.nih.gov/insidethecell/) - a science education booklet by National Institutes of Health, in PDF and ePub. • Cells Alive! (http://www.cellsalive.com/) • Cell Biology (http://www.biology.arizona.edu/cell_bio/cell_bio.html) in "The Biology Project" of University of Arizona. • Centre of the Cell online (http://www.centreofthecell.org/) • The Image & Video Library of The American Society for Cell Biology (http://cellimages.ascb.org/), a collection of peer-reviewed still images, video clips and digital books that illustrate the structure, function and biology of the cell. • HighMag Blog (http://highmagblog.blogspot.com/), still images of cells from recent research articles. • New Microscope Produces Dazzling 3D Movies of Live Cells (http://www.hhmi.org/news/betzig20110304. html), March 4, 2011 - Howard Hughes Medical Institute. • WormWeb.org: Interactive Visualization of the C. elegans Cell lineage (http://wormweb.org/celllineage) Visualize the entire cell lineage tree of the nematode C. elegans

Cells

Textbooks • Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). Molecular Biology of the Cell (http://www. ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.TOC&depth=2) (4th ed.). Garland. ISBN0-8153-3218-1. • Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipurksy SL, Darnell J (2004). Molecular Cell Biology (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.TOC) (5th ed.). WH Freeman: New York, NY. ISBN978-0-7167-4366-8. • Cooper GM (2000). The cell: a molecular approach (http://www.ncbi.nlm.nih.gov/books/bv. fcgi?rid=cooper.TOC&depth=2) (2nd ed.). Washington, D.C: ASM Press. ISBN0-87893-102-3.

Water Water is a chemical compound with the chemical formula H2O. A water molecule contains one oxygen and two hydrogen atoms connected by covalent bonds. Water is a liquid at standard ambient temperature and pressure, but it often co-exists on Earth with its solid state, ice, and gaseous state (water vapor or steam). Water also exists in a liquid crystal state near hydrophilic surfaces.[1][2] Water covers 71% of the Earth's surface,[3] Water in three states: liquid, solid (ice), and (invisible) water vapor in the air. and is vital for all known forms of life.[4] On Clouds are accumulations of water droplets, condensed from vapor-saturated air. Earth, 96.5% of the planet's water is found in oceans, 1.7% in groundwater, 1.7% in glaciers and the ice caps of Antarctica and Greenland, a small fraction in other large water bodies, and 0.001% in the air as vapor, clouds (formed of solid and liquid water particles suspended in air), and precipitation.[][5] Only 2.5% of the Earth's water is freshwater, and 98.8% of that water is in ice and groundwater. Less than 0.3% of all freshwater is in rivers, lakes, and the atmosphere, and an even smaller amount of the Earth's freshwater (0.003%) is contained within biological bodies and manufactured products.[] Water on Earth moves continually through the hydrological cycle of evaporation and transpiration (evapotranspiration), condensation, precipitation, and runoff, usually reaching the sea. Evaporation and transpiration contribute to the precipitation over land. Safe drinking water is essential to humans and other lifeforms even though it provides no calories or organic nutrients. Access to safe drinking water has improved over the last decades in almost every part of the world, but approximately one billion people still lack access to safe water and over 2.5 billion lack access to adequate sanitation.[] There is a clear correlation between access to safe water and GDP per capita.[6] However, some observers have estimated that by 2025 more than half of the world population will be facing water-based vulnerability.[7] A recent report (November 2009) suggests that by 2030, in some developing regions of the world, water demand will exceed supply by 50%.[8] Water plays an important role in the world economy, as it functions as a solvent for a wide variety of chemical substances and facilitates industrial cooling and transportation. Approximately 70% of the fresh water used by humans goes to agriculture.[]

Water

Chemical and physical properties Water is the chemical substance with chemical formula H2O: one molecule of water has two hydrogen atoms covalently bonded to a single oxygen atom. Water appears in nature in all three common states of matter (solid, liquid, and gas) and may take many different forms on Earth: water vapor and clouds in the sky; seawater in the oceans; icebergs in the polar oceans; glaciers and rivers in the mountains; and the liquid in aquifers in the ground. The major chemical and physical properties of water are:

Model of hydrogen bonds (1) between molecules of water

• Water is a liquid at standard temperature and pressure. It is tasteless and odorless. The intrinsic colour of water and ice is a very slight blue hue, although both appear colorless in small quantities. Water vapour is essentially invisible as a gas.[9] • Water is transparent in the visible electromagnetic spectrum. Thus aquatic plants can live in water because sunlight can reach them. Infrared light is strongly absorbed by the hydrogen-oxygen or OH bonds.

Impact from a water drop causes an upward "rebound" jet surrounded by circular capillary waves.

• Since the water molecule is not linear and the oxygen atom has a higher electronegativity than hydrogen atoms, it carries a slight negative charge, whereas the hydrogen atoms are slightly positive. As a result, water is a polar molecule with an electrical dipole moment. Water also can form an unusually large number of intermolecular hydrogen bonds (four) for a molecule of its size. These factors lead to strong attractive forces between molecules of water, giving rise to water's high surface tension[10] and capillary forces. The capillary action refers to the tendency of water to move up a narrow tube against the force of gravity. This property is relied upon by all vascular plants, such as trees.[11] • Water is a good polar solvent and is often referred to as the universal solvent. Substances that dissolve in water, e.g., salts, sugars, acids, alkalis, and some gases – especially oxygen, carbon dioxide (carbonation) are known as hydrophilic (water-loving) substances, while those that are immiscible with water (e.g., fats and oils), are known as hydrophobic (water-fearing) substances. • Most of the major components in cells (proteins, DNA and polysaccharides) are also dissolved in water.

Snowflakes by Wilson Bentley, 1902

• Pure water has a low electrical conductivity, but this increases with the dissolution of a small amount of ionic material such as sodium chloride.

• The boiling point of water (and all other liquids) is dependent on the barometric pressure. For example, on the top of Mt. Everest water

Water

23 boils at 68 °C (154°F), compared to 100 °C (212°F) at sea level. Conversely, water deep in the ocean near geothermal vents can reach temperatures of hundreds of degrees and remain liquid.

• At 4181.3 J/(kg·K), water has a high specific heat capacity, as well as a high heat of vaporization (40.65 kJ·mol−1), both of which are a result of the extensive hydrogen bonding between its molecules. These two unusual properties allow water to moderate Earth's climate by buffering large fluctuations in temperature. • The maximum density of water occurs at 3.98 °C (39.16°F).[12] It has the anomalous property of becoming less dense, not more, when it is cooled to its solid form, ice. During freezing, the 'open structure' of ice is gradually broken and molecules enter cavities in ice-like structure of low temperature water. There are two competing effects: 1) Increasing volume of normal liquid and 2) Decrease overall volume of the liquid. Between 0 and 3.98°C, the second effect will cancel off the first effect so the net effect is shrinkage of volume with increasing temperature.[13] It expands to occupy 9% greater volume in this solid state, which accounts for the fact of ice floating on liquid water, as in icebergs.

Dew drops adhering to a spider web

• The density of liquid water is 1,000kg/m3 (62.43lb/cu ft) at 4°C. Ice has a density of 917kg/m3 (57.25lb/cu ft). Capillary action of water compared to mercury

• Water is miscible with many liquids, such as ethanol, in all proportions, forming a single homogeneous liquid. On the other hand, water and most oils are immiscible, usually forming layers according to increasing density from the top. As a gas, water vapor is completely miscible with air. • Water forms an azeotrope with many other solvents. • Water can be split by electrolysis into hydrogen and oxygen. • As an oxide of hydrogen, water is formed when hydrogen or hydrogen-containing compounds burn or react with oxygen or oxygen-containing compounds. Water is not a fuel, it is an end-product of the combustion of hydrogen. The energy required to split water into hydrogen and oxygen by electrolysis or any other means is greater than the energy that can be collected when the hydrogen and oxygen recombine.[14]

ADR label for transporting goods dangerously reactive with water

• Elements which are more electropositive than hydrogen such as lithium, sodium, calcium, potassium and caesium displace hydrogen from water, forming hydroxides. Being a flammable gas, the hydrogen given off is dangerous and the reaction of water with the more electropositive of these elements may be violently explosive.

Water

Taste and odor Water can dissolve many different substances, giving it varying tastes and odors. Humans and other animals have developed senses that enable them to evaluate the potability of water by avoiding water that is too salty or putrid. The taste of spring water and mineral water, often advertised in marketing of consumer products, derives from the minerals dissolved in it. However, pure H2O is tasteless and odorless. The advertised purity of spring and mineral water refers to absence of toxins, pollutants and microbes, not the absence of naturally occurring minerals.

Distribution in nature In the universe Much of the universe's water is produced as a byproduct of star formation. When stars are born, their birth is accompanied by a strong outward wind of gas and dust. When this outflow of material eventually impacts the surrounding gas, the shock waves that are created compress and heat the gas. The water observed is quickly produced in this warm dense gas.[15] On 22 July 2011 a report described the discovery of a gigantic cloud of water vapor containing "140 trillion times more water than all of Earth's oceans combined" around a quasar located 12 billion light years from Earth. According to the researchers, the "discovery shows that water has been prevalent in the universe for nearly its entire existence".[][] Water has been detected in interstellar clouds within our galaxy, the Milky Way. Water probably exists in abundance in other galaxies, too, because its components, hydrogen and oxygen, are among the most abundant elements in the universe. Interstellar clouds eventually condense into solar nebulae and solar systems such as ours. Water vapor is present in • • • • • • • •

Atmosphere of Mercury: 3.4%, and large amounts of water in Mercury's exosphere[] Atmosphere of Venus: 0.002% Earth's atmosphere: ~0.40% over full atmosphere, typically 1–4% at surface Atmosphere of Mars: 0.03% Atmosphere of Jupiter: 0.0004% Atmosphere of Saturn – in ices only Enceladus (moon of Saturn): 91% exoplanets known as HD 189733 b[16] and HD 209458 b.[17]

Liquid water is present on • Earth: 71% of surface • Europa: 100km deep subsurface ocean Strong evidence suggests that liquid water is present just under the surface of Saturn's moon Enceladus. Water ice is present on • • • • • • •

Earth – mainly as ice sheets polar ice caps on Mars Moon Titan Europa Saturn's rings[] Enceladus

• Pluto and Charon[] • Comets and comet source populations (Kuiper belt and Oort cloud objects).

Water

Recent evidence points to the existence of water ice at the poles of Mercury.[18] Water ice may also be present on Ceres and Tethys. Water and other volatiles probably comprise much of the internal structures of Uranus and Neptune and the water in the deeper layers may be in the form of ionic water in which the molecules break down into a soup of hydrogen and oxygen ions, and deeper down as superionic water in which the oxygen crystallises but the hydrogen ions float around freely within the oxygen lattice.[19] Some of the Moon's minerals contain water molecules. For instance, in 2008 a laboratory device which ejects and identifies particles found small amounts of the compound in the inside of volcanic rock brought from Moon to Earth by the Apollo 15 crew in 1971.[20] NASA reported the detection of water molecules by NASA's Moon Mineralogy Mapper aboard the Indian Space Research Organization's Chandrayaan-1 spacecraft in September 2009.[21]

Water and habitable zone The existence of liquid water, and to a lesser extent its gaseous and solid forms, on Earth are vital to the existence of life on Earth as we know it. The Earth is located in the habitable zone of the solar system; if it were slightly closer to or farther from the Sun (about 5%, or about 8 million kilometers), the conditions which allow the three forms to be present simultaneously would be far less likely to exist.[22][23] Earth's gravity allows it to hold an atmosphere. Water vapor and carbon dioxide in the atmosphere provide a temperature buffer (greenhouse effect) which helps maintain a relatively steady surface temperature. If Earth were smaller, a thinner atmosphere would allow temperature extremes, thus preventing the accumulation of water except in polar ice caps (as on Mars). The surface temperature of Earth has been relatively constant through geologic time despite varying levels of incoming solar radiation (insolation), indicating that a dynamic process governs Earth's temperature via a combination of greenhouse gases and surface or atmospheric albedo. This proposal is known as the Gaia hypothesis. The state of water on a planet depends on ambient pressure, which is determined by the planet's gravity. If a planet is sufficiently massive, the water on it may be solid even at high temperatures, because of the high pressure caused by gravity, as it was observed on exoplanets Gliese 436 b[24] and GJ 1214 b.[25] There are various theories about origin of water on Earth.

On Earth Hydrology is the study of the movement, distribution, and quality of water throughout the Earth. The study of the distribution of water is hydrography. The study of the distribution and movement of groundwater is hydrogeology, of glaciers is glaciology, of inland waters is limnology and distribution of oceans is oceanography. Ecological processes with hydrology are in focus of ecohydrology. The collective mass of water found on, under, and over the surface of a planet is called the hydrosphere. Earth's

A graphical distribution of the locations of water on Earth.

Water

approximate water volume (the total water supply of the world) is 1,338,000,000km3 (321,000,000mi3).[] Liquid water is found in bodies of water, such as an ocean, sea, lake, river, stream, canal, pond, or puddle. The majority of water on Earth is sea water. Water is also present in the atmosphere in solid, liquid, and vapor states. It also exists as groundwater in aquifers. Water is important in many geological processes. Groundwater is present in most rocks, and the pressure of this groundwater affects patterns of faulting. Water in the mantle is responsible for the melt that produces volcanoes at subduction zones. On the surface of the Earth, water is important in both chemical and physical weathering processes. Water and, to a lesser but still significant extent, ice, are also responsible for a large amount of sediment transport that occurs on the surface of the earth. Deposition of transported sediment forms many types of sedimentary rocks, which make up the geologic record of Earth history.

Water covers 71% of the Earth's surface; the oceans contain 96.5% of the Earth's water. The Antarctic ice sheet, which contains 61% of all fresh water on Earth, is visible at the bottom. Condensed atmospheric water can be seen as clouds, contributing to the Earth's albedo.

Water cycle The water cycle (known scientifically as the hydrologic cycle) refers to the continuous exchange of water within the hydrosphere, between the atmosphere, soil water, surface water, groundwater, and plants. Water moves perpetually through each of these regions in the water cycle consisting of following transfer processes: • evaporation from oceans and other water bodies into the air and transpiration from land plants and animals into air.

Water cycle

• precipitation, from water vapor condensing from the air and falling to earth or ocean. • runoff from the land usually reaching the sea. Most water vapor over the oceans returns to the oceans, but winds carry water vapor over land at the same rate as runoff into the sea, about 47Tt per year. Over land, evaporation and transpiration contribute another 72Tt per year. Precipitation, at a rate of 119 Tt per year over land, has several forms: most commonly rain, snow, and hail, with some contribution from fog and dew.[26] Dew is small drops of water that are condensed when a high density of water vapor meets a cool surface. Dew usually form in the morning when the temperature is the lowest, just before sunrise and when the temperature of the earth's surface starts to increase.[27] Condensed water in the air may also refract sunlight to produce rainbows.

Water Water runoff often collects over watersheds flowing into rivers. A mathematical model used to simulate river or stream flow and calculate water quality parameters is hydrological transport model. Some of water is diverted to irrigation for agriculture. Rivers and seas offer opportunity for travel and commerce. Through erosion, runoff shapes the environment creating river valleys and deltas which provide rich soil and level ground for the establishment of population centers. A flood occurs when an area of land, usually low-lying, is covered with water. It is when a river overflows its banks or flood from the sea. A drought is an extended period of months or years when a region notes a deficiency in its water supply. This occurs when a region receives consistently below average precipitation.

Fresh water storage

The Bay of Fundy at high tide (left) and low tide (right) Some runoff water is trapped for periods of time, for example in lakes. At high altitude, during winter, and in the far north and south, snow collects in ice caps, snow pack and glaciers. Water also infiltrates the ground and goes into aquifers. This groundwater later flows back to the surface in springs, or more spectacularly in hot springs and geysers. Groundwater is also extracted artificially in wells. This water storage is important, since clean, fresh water is essential to human and other land-based life. In many parts of the world, it is in short supply.

Sea water Sea water contains about 3.5% salt on average, plus smaller amounts of other substances. The physical properties of sea water differ from fresh water in some important respects. It freezes at a lower temperature (about −1.9 °C) and its density increases with decreasing temperature to the freezing point, instead of reaching maximum density at a temperature above freezing. The salinity of water in major seas varies from about 0.7% in the Baltic Sea to 4.0% in the Red Sea.

Tides Tides are the cyclic rising and falling of local sea levels caused by the tidal forces of the Moon and the Sun acting on the oceans. Tides cause changes in the depth of the marine and estuarine water bodies and produce oscillating currents known as tidal streams. The changing tide produced at a given location is the result of the changing positions of the Moon and Sun relative to the Earth coupled with the effects of Earth rotation and the local bathymetry. The strip of seashore that is submerged at high tide and exposed at low tide, the intertidal zone, is an important ecological product of ocean tides.

Water

Effects on life From a biological standpoint, water has many distinct properties that are critical for the proliferation of life that set it apart from other substances. It carries out this role by allowing organic compounds to react in ways that ultimately allow replication. All known forms of life depend on water. Water is vital both as a solvent in which many of the body's solutes dissolve and as an essential part of many metabolic processes within the body. Metabolism is the sum total of anabolism and catabolism. In anabolism, water is removed from molecules (through energy requiring enzymatic chemical reactions) in order to grow larger molecules (e.g. starches, triglycerides and proteins for storage of fuels and information). In catabolism, water is used to break bonds in order to generate smaller molecules (e.g. glucose, fatty acids and amino acids to be used for fuels for energy use or other purposes). Without water, these particular metabolic processes could not exist.

Overview of photosynthesis and respiration. Water (at right), together with carbon dioxide (CO2), form oxygen and organic compounds (at left), which can be respired to water and (CO2).

Water is fundamental to photosynthesis and respiration. Photosynthetic cells use the sun's energy to split off water's hydrogen from oxygen. Hydrogen is combined with CO2 (absorbed from air or water) to form glucose and release oxygen. All living cells use such fuels and oxidize the hydrogen and carbon to capture the sun's energy and reform water and CO2 in the process (cellular respiration). Water is also central to acid-base neutrality and enzyme function. An acid, a hydrogen ion (H+, that is, a proton) donor, can be neutralized by a base, a proton acceptor such as hydroxide ion (OH−) to form water. Water is considered to be neutral, with a pH (the negative log of the hydrogen ion concentration) of 7. Acids have pH values less than 7 while bases have values greater than 7.

Aquatic life forms Earth surface waters are filled with life. The earliest life forms appeared in water; nearly all fish live exclusively in water, and there are many types of marine mammals, such as dolphins and whales. Some kinds of animals, such as amphibians, spend portions of their lives in water and portions on land. Plants such as kelp and algae grow in the water and are the basis for some underwater ecosystems. Plankton is generally the foundation of the ocean food chain. Aquatic vertebrates must obtain oxygen to survive, and they do so in various ways. Fish have gills instead of lungs, although some species of fish, such as the lungfish, have both. Marine mammals, such as dolphins, whales, otters, and seals need to surface periodically to breathe air. Some amphibians are able to absorb oxygen through their skin. Invertebrates exhibit a wide range of modifications to survive in poorly oxygenated waters including breathing tubes (see insect and mollusc siphons) and gills (Carcinus). However as invertebrate life evolved in an aquatic habitat most have little or no specialisation for respiration in water.

Some of the biodiversity of a coral reef

Water

Some marine diatoms – a key phytoplankton group

Effects on human civilization Civilization has historically flourished around rivers and major waterways; Mesopotamia, the so-called cradle of civilization, was situated between the major rivers Tigris and Euphrates; the ancient society of the Egyptians depended entirely upon the Nile. Large metropolises like Rotterdam, London, Montreal, Paris, New York City, Buenos Aires, Shanghai, Tokyo, Chicago, and Hong Kong owe their success in part to their easy accessibility via water and the resultant expansion of trade. Islands with safe water ports, like Singapore, have flourished for the same reason. In places such as North Africa and the Middle East, where water is more scarce, access to clean drinking water was and is a major factor in human development.

Water fountain

Health and pollution Water fit for human consumption is called drinking water or potable water. Water that is not potable may be made potable by filtration or distillation, or by a range of other methods. Water that is not fit for drinking but is not harmful for humans when used for swimming or bathing is called by various names other than potable or drinking water, and is sometimes called safe water, or "safe for bathing". Chlorine is a skin and mucous membrane irritant that is used to make water safe for bathing or drinking. Its use is highly technical and is usually monitored by government regulations An environmental science program - a student (typically 1 part per million (ppm) for drinking water, and 1–2 ppm of from Iowa State University sampling water chlorine not yet reacted with impurities for bathing water). Water for bathing may be maintained in satisfactory microbiological condition using chemical disinfectants such as chlorine or ozone or by the use of ultraviolet light. In the USA, non-potable forms of wastewater generated by humans may be referred to as greywater, which is treatable and thus easily able to be made potable again, and blackwater, which generally contains sewage and other forms of waste which require further treatment in order to be made reusable. Greywater composes 50–80% of residential wastewater generated by a household's sanitation equipment (sinks, showers and kitchen runoff, but not toilets, which generate blackwater.) These terms may have different meanings in other countries and cultures.

Water

This natural resource is becoming scarcer in certain places, and its availability is a major social and economic concern. Currently, about a billion people around the world routinely drink unhealthy water. Most countries accepted the goal of halving by 2015 the number of people worldwide who do not have access to safe water and sanitation during the 2003 G8 Evian summit.[28] Even if this difficult goal is met, it will still leave more than an estimated half a billion people without access to safe drinking water and over a billion without access to adequate sanitation. Poor water quality and bad sanitation are deadly; some five million deaths a year are caused by polluted drinking water. The World Health Organization estimates that safe water could prevent 1.4 million child deaths from diarrhea each year.[29] Water, however, is not a finite resource, but rather re-circulated as potable water in precipitation in quantities many degrees of magnitude higher than human consumption. Therefore, it is the relatively small quantity of water in reserve in the earth (about 1% of our drinking water supply, which is replenished in aquifers around every 1 to 10 years), that is a non-renewable resource, and it is, rather, the distribution of potable and irrigation water which is scarce, rather than the actual amount of it that exists on the earth. Water-poor countries use importation of goods as the primary method of importing water (to leave enough for local human consumption), since the manufacturing process uses around 10 to 100 times products' masses in water. In the developing world, 90% of all wastewater still goes untreated into local rivers and streams.[30] Some 50 countries, with roughly a third of the world's population, also suffer from medium or high water stress, and 17 of these extract more water annually than is recharged through their natural water cycles.[31] The strain not only affects surface freshwater bodies like rivers and lakes, but it also degrades groundwater resources.

Human uses Agriculture The most important use of water in agriculture is for irrigation, which is a key component to produce enough food. Irrigation takes up to 90% of water withdrawn in some developing countries[32] and significant proportions in more economically developed countries (United States, 30% of freshwater usage is for irrigation).[33] It takes around 3,000 litres of water, converted from liquid to vapour, to produce enough food to satisfy one person's daily dietary need. This is a considerable amount, when compared to that required for drinking, which is between two and five litres. To produce food for the 6.5 billion or so people who inhabit the planet today requires the water that would fill a canal ten metres deep, 100 metres wide and 7.1 million kilometres long – that's enough to circle the globe 180 times.

Water distribution in subsurface drip irrigation.

Fifty years ago, the common perception was that water was an infinite resource. At this time, there were fewer than half the current number of people on the planet. People were not as wealthy as today, consumed fewer calories and ate less meat, so less water was needed to produce their food. They required a third of the volume of water we presently take from rivers. Today, the competition for the fixed amount of water resources is much more intense, giving rise to the concept of peak Irrigation of field crops water.[34] This is because there are now nearly seven billion people on the planet, their consumption of water-thirsty meat and vegetables is rising, and there is increasing competition for water from industry, urbanisation and biofuel crops. In future, even more water will be needed to produce food because the Earth's population is forecast to rise to 9 billion by 2050.[35]

Water An additional 2.5 or 3 billion people, choosing to eat fewer cereals and more meat and vegetables could add an additional five million kilometres to the virtual canal mentioned above. An assessment of water management in agriculture was conducted in 2007 by the International Water Management Institute in Sri Lanka to see if the world had sufficient water to provide food for its growing population.[36] It assessed the current availability of water for agriculture on a global scale and mapped out locations suffering from water scarcity. It found that a fifth of the world's people, more than 1.2 billion, live in areas of physical water scarcity, where there is not enough water to meet all demands. A further 1.6 billion people live in areas experiencing economic water scarcity, where the lack of investment in water or insufficient human capacity make it impossible for authorities to satisfy the demand for water. The report found that it would be possible to produce the food required in future, but that continuation of today's food production and environmental trends would lead to crises in many parts of the world. To avoid a global water crisis, farmers will have to strive to increase productivity to meet growing demands for food, while industry and cities find ways to use water more efficiently.[37] As a scientific standard On 7 April 1795, the gram was defined in France to be equal to "the absolute weight of a volume of pure water equal to a cube of one hundredth of a meter, and to the temperature of the melting ice."[38] For practical purposes though, a metallic reference standard was required, one thousand times more massive, the kilogram. Work was therefore commissioned to determine precisely the mass of one liter of water. In spite of the fact that the decreed definition of the gram specified water at 0°C— a highly reproducible temperature— the scientists chose to redefine the standard and to perform their measurements at the temperature of highest water density, which was measured at the time as 4 °C (39°F).[39] The Kelvin temperature scale of the SI system is based on the triple point of water, defined as exactly 273.16K or 0.01°C. The scale is an absolute temperature scale with the same increment as the Celsius temperature scale, which was originally defined according the boiling point (set to 100°C) and melting point (set to 0°C) of water. Natural water consists mainly of the isotopes hydrogen-1 and oxygen-16, but there is also small quantity of heavier isotopes such as hydrogen-2 (deuterium). The amount of deuterium oxides or heavy water is very small, but it still affects the properties of water. Water from rivers and lakes tends to contain less deuterium than seawater. Therefore, standard water is defined in the Vienna Standard Mean Ocean Water specification. For drinking The human body contains from 55% to 78% water, depending on body size.[40] To function properly, the body requires between one and seven liters of water per day to avoid dehydration; the precise amount depends on the level of activity, temperature, humidity, and other factors. Most of this is ingested through foods or beverages other than drinking straight water. It is not clear how much water intake is needed by healthy people, though most advocates agree that approximately 2 liters (6 to 7 glasses) of water daily is the minimum to maintain proper hydration.[41] Medical literature favors a lower consumption, typically A young girl drinking bottled water 1 liter of water for an average male, excluding extra requirements due to fluid loss from exercise or warm weather.[] For those who have healthy kidneys, it is rather difficult to drink too much water, but (especially in warm humid weather and while exercising) it is dangerous to drink too little. People can drink far more water than

Water

necessary while exercising, however, putting them at risk of water intoxication (hyperhydration), which can be fatal.[42][43] The popular claim that "a person should consume eight glasses of water per day" seems to have no real basis in science.[44] Similar misconceptions concerning the effect of water on weight loss and constipation have also been dispelled.[45] Water availability: fraction of population using improved water sources by country.

An original recommendation for water intake in 1945 by the Food and Nutrition Board of the United States National Research Council read: "An ordinary standard for diverse persons is 1 milliliter for each calorie of food. Most of this quantity is contained in prepared foods."[46] The latest dietary reference intake report by the United States National Research Council in general recommended (including food sources): 3.7 liters for men and 2.7 liters of water total for women.[47] Specifically, pregnant and breastfeeding women need additional fluids to stay hydrated. The Institute of Medicine (U.S.) recommends that, on average, men consume 3.0 liters and women 2.2 liters; pregnant women should increase intake to 2.4 liters (10 cups) and breastfeeding women should get 3 liters (12 cups), since an especially large amount Hazard symbol for non-potable water of fluid is lost during nursing.[48] Also noted is that normally, about 20% of water intake comes from food, while the rest comes from drinking water and beverages (caffeinated included). Water is excreted from the body in multiple forms; through urine and feces, through sweating, and by exhalation of water vapor in the breath. With physical exertion and heat exposure, water loss will increase and daily fluid needs may increase as well. Humans require water with few impurities. Common impurities include metal salts and oxides, including copper, iron, calcium and lead,[49] and/or harmful bacteria, such as Vibrio. Some solutes are acceptable and even desirable for taste enhancement and to provide needed electrolytes.[50] The single largest (by volume) freshwater resource suitable for drinking is Lake Baikal in Siberia.[51] Washing The propensity of water to form solutions and emulsions is useful in various washing processes. Many industrial processes rely on reactions using chemicals dissolved in water, suspension of solids in water slurries or using water to dissolve and extract substances. Washing is also an important component of several aspects of personal body hygiene. Transportation The use of water for transportation of materials through rivers and canals as well as the international shipping lanes is an important part of the world economy.

Water

Chemical uses Water is widely used in chemical reactions as a solvent or reactant and less commonly as a solute or catalyst. In inorganic reactions, water is a common solvent, dissolving many ionic compounds. In organic reactions, it is not usually used as a reaction solvent, because it does not dissolve the reactants well and is amphoteric (acidic and basic) and nucleophilic. Nevertheless, these properties are sometimes desirable. Also, acceleration of Diels-Alder reactions by water has been observed. Supercritical water has recently been a topic of research. Oxygen-saturated supercritical water combusts organic pollutants efficiently. Heat exchange Water and steam are used as heat transfer fluids in diverse heat exchange systems, due to its availability and high heat capacity, both as a coolant and for heating. Cool water may even be naturally available from a lake or the sea. Condensing steam is a particularly efficient heating fluid because of the large heat of vaporization. A disadvantage is that water and steam are somewhat corrosive. In almost all electric power stations, water is the coolant, which vaporizes and drives steam turbines to drive generators. In the U.S., cooling power plants is the largest use of water.[33] In the nuclear power industry, water can also be used as a neutron moderator. In most nuclear reactors, water is both a coolant and a moderator. This provides something of a passive safety measure, as removing the water from the reactor also slows the nuclear reaction down – however other methods are favored for stopping a reaction and it is preferred to keep the nuclear core covered with water so as to ensure adequate cooling. Fire extinction Water has a high heat of vaporization and is relatively inert, which makes it a good fire extinguishing fluid. The evaporation of water carries heat away from the fire. It is dangerous to use water on fires involving oils and organic solvents, because many organic materials float on water and the water tends to spread the burning liquid. Use of water in fire fighting should also take into account the hazards of a steam explosion, which may occur when water is used on very hot fires in confined spaces, and of a hydrogen explosion, when substances which react with water, such as certain metals or hot carbon such as coal, charcoal, coke graphite, decompose the water, producing water gas.

Water is used for fighting wildfires.

The power of such explosions was seen in the Chernobyl disaster, although the water involved did not come from fire-fighting at that time but the reactor's own water cooling system. A steam explosion occurred when the extreme overheating of the core caused water to flash into steam. A hydrogen explosion may have occurred as a result of reaction between steam and hot zirconium.

Water

Recreation Humans use water for many recreational purposes, as well as for exercising and for sports. Some of these include swimming, waterskiing, boating, surfing and diving. In addition, some sports, like ice hockey and ice skating, are played on ice. Lakesides, beaches and water parks are popular places for people to go to relax and enjoy recreation. Many find the sound and appearance of flowing water to be calming, and fountains and other water features are popular decorations. Some keep fish and other life in aquariums or ponds for show, fun, and companionship. Humans also use water for snow sports i.e. skiing, sledding, snowmobiling or snowboarding, which requires the water to be frozen.

Grand Anse Beach, St. George's, Grenada, West Indies.

Water industry The water industry provides drinking water and wastewater services (including sewage treatment) to households and industry. Water supply facilities include water wells cisterns for rainwater harvesting, water supply network, water purification facilities, water tanks, water towers, water pipes including old aqueducts. Atmospheric water generators are in development. Drinking water is often collected at springs, extracted from artificial borings (wells) in the ground, or pumped from lakes and rivers. Building more wells in adequate places is thus a possible way to produce more water, assuming the aquifers can supply an adequate flow. Other water sources include rainwater collection. Water may require purification for human consumption. This may involve removal of undissolved substances, dissolved substances and harmful microbes. Popular methods are filtering with sand which only removes undissolved material, while chlorination and boiling kill harmful microbes. Distillation does all three functions. More advanced techniques exist, such as reverse osmosis. Desalination of abundant seawater is a more expensive solution used in coastal arid climates.

A water-carrier in India, 1882. In many places where running water is not available, water has to be transported by people.

The distribution of drinking water is done through municipal water systems, tanker delivery or as bottled water. Governments in many countries have programs to distribute water to the needy at no charge. Reducing usage by using drinking (potable) water only for human consumption is another option. In some cities such as Hong Kong, sea water is extensively used for flushing toilets citywide in order to conserve fresh water resources.

A manual water pump in China

Polluting water may be the biggest single misuse of water; to the extent that a pollutant limits other uses of the water, it becomes a waste of the resource, regardless of benefits to the polluter. Like other types of pollution, this does not enter standard accounting of market costs,

Water

being conceived as externalities for which the market cannot account. Thus other people pay the price of water pollution, while the private firms' profits are not redistributed to the local population victim of this pollution. Pharmaceuticals consumed by humans often end up in the waterways and can have detrimental effects on aquatic life if they bioaccumulate and if they are not biodegradable. Wastewater facilities are storm sewers and wastewater treatment plants. Another way to remove pollution from surface runoff water is bioswale.

Water purification facility

Industrial applications Water is used in power generation. Hydroelectricity is electricity obtained from hydropower. Hydroelectric power comes from water driving a water turbine connected to a generator. Hydroelectricity is a low-cost, non-polluting, renewable energy source. The energy is supplied by the motion of water. Typically a dam is constructed on a river, creating an artificial lake behind it. Water flowing out of the lake is forced through turbines that turn generators.

Three Gorges Dam is the largest hydro-electric power station. Pressurized water is used in water blasting and water jet cutters. Also, very high pressure water guns are used for precise cutting. It works very well, is relatively safe, and is not harmful to the environment. It is also used in the cooling of machinery to prevent overheating, or prevent saw blades from overheating. Water is also used in many industrial processes and machines, such as the steam turbine and heat exchanger, in addition to its use as a chemical solvent. Discharge of untreated water from industrial uses is pollution. Pollution includes discharged solutes (chemical pollution) and discharged coolant water (thermal pollution). Industry requires pure water for many applications and utilizes a variety of purification techniques both in water supply and discharge.

Water Food processing Water plays many critical roles within the field of food science. It is important for a food scientist to understand the roles that water plays within food processing to ensure the success of their products. Solutes such as salts and sugars found in water affect the physical properties of water. The boiling and freezing points of water are affected by solutes, as well as air pressure, which is in turn affected by altitude. Water boils at lower temperatures with the lower air pressure which occurs at higher elevations. One mole of sucrose (sugar) per kilogram of water raises the boiling point of water by 0.51 °C, and one Water can be used to cook foods such as noodles. mole of salt per kg raises the boiling point by 1.02 °C; similarly, increasing the number of dissolved particles lowers water's freezing point.[] Solutes in water also affect water activity which affects many chemical reactions and the growth of microbes in food.[] Water activity can be described as a ratio of the vapor pressure of water in a solution to the vapor pressure of pure water.[] Solutes in water lower water activity. This is important to know because most bacterial growth ceases at low levels of water activity.[] Not only does microbial growth affect the safety of food but also the preservation and shelf life of food. Water hardness is also a critical factor in food processing. It can dramatically affect the quality of a product as well as playing a role in sanitation. Water hardness is classified based on the amounts of removable calcium carbonate salt it contains per gallon. Water hardness is measured in grains; 0.064 g calcium carbonate is equivalent to one grain of hardness.[] Water is classified as soft if it contains 1 to 4 grains, medium if it contains 5 to 10 grains and hard if it contains 11 to 20 grains. Wikipedia:Vagueness [] The hardness of water may be altered or treated by using a chemical ion exchange system. The hardness of water also affects its pH balance which plays a critical role in food processing. For example, hard water prevents successful production of clear beverages. Water hardness also affects sanitation; with increasing hardness, there is a loss of effectiveness for its use as a sanitizer.[] Boiling, steaming, and simmering are popular cooking methods that often require immersing food in water or its gaseous state, steam. Water is also used for dishwashing.

Water

Water law, water politics and water crisis Water politics is politics affected by water and water resources. For this reason, water is a strategic resource in the globe and an important element in many political conflicts. It causes health impacts and damage to biodiversity. 1.6 billion people have gained access to a safe water source since 1990.[52] The proportion of people in developing countries with access to safe water is calculated to have improved from 30% in 1970[] to 71% in 1990, 79% in 2000 and 84% in 2004. This trend is projected to continue.[] To halve, by 2015, the proportion of people without sustainable access to safe drinking water is one of the Millennium Development Goals. This goal is projected to be reached.

An estimate of the share of people in developing countries with access to potable water 1970–2000

A 2006 United Nations report stated that "there is enough water for everyone", but that access to it is hampered by mismanagement and corruption.[53] In addition, global initiatives to improve the efficiency of aid delivery, such as the Paris Declaration on Aid Effectiveness, have not been taken up by water sector donors as effectively as they have in education and health, potentially leaving multiple donors working on overlapping projects and recipient governments without empowerment to act.[54] The authors of the 2007 Comprehensive Assessment of Water Management in Agriculture cited poor governance as one reason for some forms of water scarcity. Water governance is the set of formal and informal processes through which decisions related to water management are made. Good water governance is primarily about knowing what processes work best in a particular physical and socioeconomic context. Mistakes have sometimes been made by trying to apply 'blueprints' that work in the developed world to developing world locations and contexts. The Mekong river is one example; a review by the International Water Management Institute of policies in six countries that rely on the Mekong river for water found that thorough and transparent cost-benefit analyses and environmental impact assessments were rarely undertaken. They also discovered that Cambodia's draft water law was much more complex than it needed to be.[55] The UN World Water Development Report (WWDR, 2003) from the World Water Assessment Program indicates that, in the next 20 years, the quantity of water available to everyone is predicted to decrease by 30%. 40% of the world's inhabitants currently have insufficient fresh water for minimal hygiene. More than 2.2 million people died in 2000 from waterborne diseases (related to the consumption of contaminated water) or drought. In 2004, the UK charity WaterAid reported that a child dies every 15 seconds from easily preventable water-related diseases; often this means lack of sewage disposal; see toilet. Organizations concerned with water protection include International Water Association (IWA), WaterAid, Water 1st, American Water Resources Association [56]. The International Water Management Institute undertakes projects with the aim of using effective water management to reduce poverty. Water related conventions are United Nations Convention to Combat Desertification (UNCCD), International Convention for the Prevention of Pollution from Ships, United Nations Convention on the Law of the Sea and Ramsar Convention. World Day for Water takes place on 22 March and World Ocean Day on 8 June.

Water Water used in the production of a good or service is virtual water.

In culture Religion Water is considered a purifier in most religions. Major faiths that incorporate ritual washing (ablution) include Christianity, Hinduism, Islam, Judaism, Rastafari movement, Shinto, Taoism, and Wicca. Immersion (or aspersion or affusion) of a person in water is a central sacrament of Christianity (where it is called baptism); it is also a part of the practice of other religions, including Islam (Ghusl), Judaism (mikvah) and Sikhism (Amrit Sanskar). In addition, a ritual bath in pure water is performed for the dead in many religions including Islam and Judaism. In Islam, the five daily prayers can be done in most cases after completing washing certain parts of the body using clean water (wudu), unless water is unavailable (see Tayammum). In Shinto, water is used in almost all rituals to cleanse a person or an area (e.g., in the ritual of misogi). Water is mentioned numerous times in the Bible, for example: "The earth was formed out of water and by water" (NIV). In the Qur'an it is stated that "Living things are made of water" and it is often used to describe paradise.

Philosophy The Ancient Greek philosopher Empedocles held that water is one of the four classical elements along with fire, earth and air, and was regarded as the ylem, or basic substance of the universe. Water was considered cold and moist. In the theory of the four bodily humors, water was associated with phlegm. The classical element of Water was also one of the five elements in traditional Chinese philosophy, along with earth, fire, wood, and metal. Water is also taken as a role model in some parts of traditional and popular Asian philosophy. James Legge's 1891 translation of the Dao De Jing states "The highest excellence is like (that of) water. The excellence of water appears in its benefiting all things, and in its occupying, without striving (to the contrary), the low place which all men dislike. Hence (its way) is near to (that of) the Tao" and "There is nothing in the world more soft and weak than water, and yet for attacking things that are firm and strong there is nothing that can take precedence of it—for there is nothing (so effectual) for which it can be changed."[57]

Literature Water is used in literature as a symbol of purification. Examples include the critical importance of a river in As I Lay Dying by William Faulkner and the drowning of Ophelia in Hamlet. Sherlock Holmes held that "From a drop of water, a logician could infer the possibility of an Atlantic or a Niagara without having seen or heard of one or the other."[58]

References [5] Water Vapor in the Climate System (http:/ / www. agu. org/ sci_soc/ mockler. html), Special Report, [AGU], December 1995 (linked 4/2007). Vital Water (http:/ / www. unep. org/ dewa/ assessments/ ecosystems/ water/ vitalwater/ ) UNEP. [6] "Public Services" (http:/ / www. gapminder. org/ videos/ gapcasts/ gapcast-9-public-services/ ), Gapminder video [11] Capillary Action– Liquid, Water, Force, and Surface – JRank Articles (http:/ / science. jrank. org/ pages/ 1182/ Capillary-Action. html) [15] Melnick, Gary, Harvard-Smithsonian Center for Astrophysics and Neufeld, David, Johns Hopkins University quoted in:

(linked 4/2007) [16] Water Found on Distant Planet (http:/ / www. time. com/ time/ health/ article/ 0,8599,1642811,00. html) July 12, 2007 By Laura Blue, Time [17] Water Found in Extrasolar Planet's Atmosphere (http:/ / www. space. com/ scienceastronomy/ 070410_water_exoplanet. html) – Space.com [18] NASA, " MESSENGER Finds New Evidence for Water Ice at Mercury's Poles (http:/ / www. nasa. gov/ mission_pages/ messenger/ media/ PressConf20121129. html)", 29 November 2012. [19] Weird water lurking inside giant planets (http:/ / www. newscientist. com/ article/ mg20727764. 500-weird-water-lurking-inside-giant-planets. html), New Scientist, 1 September 2010, Magazine issue 2776.

Water [20] Versteckt in Glasperlen: Auf dem Mond gibt es Wasser – Wissenschaft – [[Der Spiegel (http:/ / www. spiegel. de/ wissenschaft/ weltall/ 0,1518,564911,00. html)] – Nachrichten] [21] Water Molecules Found on the Moon (http:/ / science. nasa. gov/ headlines/ y2009/ 24sep_moonwater. htm), NASA, 24 September 2009 [33] Water Use in the United States (http:/ / nationalatlas. gov/ articles/ water/ a_wateruse. html), National Atlas.gov [35] United Nations Press Release POP/952, 13 March 2007. World population will increase by 2.5 billion by 2050 (http:/ / www. un. org/ News/ Press/ docs/ 2007/ pop952. doc. htm) [36] Molden, D. (Ed). Water for food, Water for life: A Comprehensive Assessment of Water Management in Agriculture. Earthscan/IWMI, 2007. [37] Chartres, C. and Varma, S. Out of water. From Abundance to Scarcity and How to Solve the World's Water Problems FT Press (USA), 2010 [38] Décret relatif aux poids et aux mesures. 18 germinal an 3 (7 avril 1795) (http:/ / smdsi. quartier-rural. org/ histoire/ 18germ_3. htm). Decree relating to the weights and measurements (in French). quartier-rural.org [39] here L'Histoire Du Mètre, La Détermination De L'Unité De Poids (http:/ / histoire. du. metre. free. fr/ fr/ index. htm). histoire.du.metre.free.fr [40] Re: What percentage of the human body is composed of water? (http:/ / www. madsci. org/ posts/ archives/ 2000-05/ 958588306. An. r. html) Jeffrey Utz, M.D., The MadSci Network [44] "Drink at least eight glasses of water a day." Really? Is there scientific evidence for "8 × 8"? (http:/ / ajpregu. physiology. org/ cgi/ content/ full/ 283/ 5/ R993) by Heinz Valdin, Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire [45] Drinking Water – How Much? (http:/ / www. factsmart. org/ h2o/ h2o. htm), Factsmart.org web site and references within [47] Dietary Reference Intakes: Water, Potassium, Sodium, Chloride, and Sulfate (http:/ / www. iom. edu/ report. asp?id=18495), Food and Nutrition Board [49] "Conquering Chemistry" 4th Ed. Published 2008 [52] The Millennium Development Goals Report (http:/ / mdgs. un. org/ unsd/ mdg/ Resources/ Static/ Products/ Progress2008/ MDG_Report_2008_En. pdf#page=44), United Nations, 2008 [53] UNESCO, (2006), Water, a shared responsibility. The United Nations World Water Development Report 2 (http:/ / unesdoc. unesco. org/ images/ 0014/ 001444/ 144409E. pdf). [54] Welle, Katharina; Evans, Barbara; Tucker, Josephine and Nicol, Alan (2008) Is water lagging behind on Aid Effectiveness? (http:/ / www. odi. org. uk/ resources/ download/ 1894. pdf) [55] Water governance (http:/ / www. iwmi. cgiar. org/ Publications/ Water_Issue_Briefs/ index. aspx), Water Issue Brief, Issue 5, 2010, IWMI [56] http:/ / www. awra. org/ [58] Arthur Conan Doyle, A Study in Scarlet, Chapter 2, "The Science of Deduction"

Further reading • Debenedetti,PG., and HE Stanley, "Supercooled and Glassy Water", Physics Today 56 (6), p.40–46 (2003). Downloadable PDF (1.9 MB) (http://polymer.bu.edu/hes/articles/ds03.pdf) • Franks, F (Ed), Water, A comprehensive treatise, Plenum Press, New York, 1972–1982 • Gleick, PH., (editor), The World's Water: The Biennial Report on Freshwater Resources. Island Press, Washington, D.C. (published every two years, beginning in 1998.) The World's Water, Island Press (http://www. worldwater.org/) • Jones, OA., JN Lester and N Voulvoulis, Pharmaceuticals: a threat to drinking water? TRENDS in Biotechnology 23(4): 163, 2005 • Journal of Contemporary Water Resources and Education (http://ucowr.org/updates/index.html) • Postel,S., Last Oasis: Facing Water Scarcity. W.W. Norton and Company, New York. 1992 • Reisner,M., Cadillac Desert: The American West and Its Disappearing Water. Penguin Books, New York. 1986. • United Nations World Water Development Report. Produced every three years. UN World Water Development Report (http://www.unesco.org/water/wwap/wwdr/)

Water

External links • • • • • •

OECD Water statistics (http://stats.oecd.org/wbos/Index.aspx?DataSetCode=ENV_WAT) The World's Water Data Page (http://www.worldwater.org/) FAO Comprehensive Water Database, AQUASTAT (http://www.fao.org/nr/water/aquastat/main/index.stm) The Water Conflict Chronology: Water Conflict Database (http://worldwater.org/conflict.html) US Geological Survey Water for Schools information (http://ga.water.usgs.gov/edu/) Portal to The World Bank's strategy, work and associated publications on water resources (http://water. worldbank.org/)

Structural Biochemistry

Nucleic acids Nucleic acid Nucleic acids are large biological molecules essential for all known forms of life. They include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Together with proteins, nucleic acids are the most important biological macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information. Nucleic acids were discovered by Friedrich Miescher in 1869.[1] Experimental studies of nucleic acids constitute a major part of modern biological and medical research, and form a foundation for genome and forensic science, as well as the biotechnology and pharmaceutical industries.[][][]

Occurrence and nomenclature[]

A comparison of the two principal nucleic acids: RNA (left) and DNA (right), showing the helices and nucleobases each employs.

The term nucleic acid is the overall name for DNA and RNA, members of a family of biopolymers,[2] and is synonymous with polynucleotide. Nucleic acids were named for their initial discovery within the nucleus, and for the presence of phosphate groups (related to phosphoric acid). Although first discovered within the nucleus of eukaryotic cells, nucleic acids are now known to be found in all life forms as well as some nonliving entities, including within bacteria, archaea, mitochondria, chloroplasts, viruses and viroids. All living cells contain both DNA and RNA (except some cells such as mature red blood cells), while viruses contain either DNA or RNA, but usually not both.[] The basic component of biological nucleic acids is the nucleotide, each of which contains a pentose sugar (ribose or deoxyribose), a phosphate group, and a nucleobase. Nucleic acids are also generated within the laboratory, through the use of enzymes[3] (DNA and RNA polymerases) and by solid-phase chemical synthesis. The chemical methods also enable the generation of altered nucleic acids that are not found in nature,[4] for example peptide nucleic acids.

Nucleic acid

Molecular composition and size[] Nucleic acids can vary in size, but are generally very large molecules. Indeed, DNA molecules are probably the largest individual molecules known. Well-studied biological nucleic acid molecules range in size from 21 nucleotides (small interfering RNA) to large chromosomes (human chromosome 1 is a single molecule that contains 247 million base pairs[5]). In most cases, naturally occurring DNA molecules are double-stranded and RNA molecules are single-stranded. There are numerous exceptions, however—some viruses have genomes made of double-stranded RNA and other viruses have single-stranded DNA genomes, and, in some circumstances, nucleic acid structures with three or four strands can form. Nucleic acids are linear polymers (chains) of nucleotides. Each nucleotide consists of three components: a purine or pyrimidine nucleobase (sometimes termed nitrogenous base or simply base), a pentose sugar, and a phosphate group. The substructure consisting of a nucleobase plus sugar is termed a nucleoside. Nucleic acid types differ in the structure of the sugar in their nucleotides - DNA contains 2'-deoxyribose while RNA contains ribose (where the only difference is the presence of a hydroxyl group). Also, the nucleobases found in the two nucleic acid types are different: adenine, cytosine, and guanine are found in both RNA and DNA, while thymine occurs in DNA and uracil occurs in RNA. The sugars and phosphates in nucleic acids are connected to each other in an alternating chain (sugar-phosphate backbone) through phosphodiester linkages.[] In conventional nomenclature, the carbons to which the phosphate groups attach are the 3'-end and the 5'-end carbons of the sugar. This gives nucleic acids directionality, and the ends of nucleic acid molecules are referred to as 5'-end and 3'-end. The nucleobases are joined to the sugars via an N-glycosidic linkage involving a nucleobase ring nitrogen (N-1 for pyrimidines and N-9 for purines) and the 1' carbon of the pentose sugar ring. Non-standard nucleosides are also found in both RNA and DNA and usually arise from modification of the standard nucleosides within the DNA molecule or the primary (initial) RNA transcript. Transfer RNA (tRNA) molecules contain a particularly large number of modified nucleosides.[6]

Topology Double-stranded nucleic acids are made up of complementary sequences, in which extensive Watson-Crick base pairing results in the a highly repeated and quite uniform double-helical three-dimensional structure.[7] In contrast, single-stranded RNA and DNA molecules are not constrained to a regular double helix, and can adopt highly complex three-dimensional structures that are based on short stretches of intramolecular base-paired sequences that include both Watson-Crick and noncanonical base pairs, as well as a wide range of complex tertiary interactions.[8] Nucleic acid molecules are usually unbranched, and may occur as linear and circular molecules. For example, bacterial chromosomes, plasmids, mitochondrial DNA and chloroplast DNA are usually circular double-stranded DNA molecules, while chromosomes of the eukaryotic nucleus are usually linear double-stranded DNA molecules.[] Most RNA molecules are linear, single-stranded molecules, but both circular and branched molecules can result from RNA splicing reactions.[]

Nucleic acid sequences One DNA or RNA molecule differs from another primarily in the sequence of nucleotides. Nucleotide sequences are of great importance in biology, since they carry the ultimate instructions that encode all biological molecules, molecular assemblies, subcellular and cellular structures, organs and organisms, and directly enable cognition, memory and behavior (See: Genetics). Enormous efforts have gone into the development of experimental methods to determine the nucleotide sequence of biological DNA and RNA molecules,[9][10] and today hundreds of millions of nucleotides are sequenced daily at genome centers and smaller laboratories worldwide.

Nucleic acid

Types of nucleic acids Deoxyribonucleic acid Deoxyribonucleic acid (/diˌɒksiˌraɪbɵ.njuːˌkleɪ.ɨk ˈæsɪd/; DNA) is a nucleic acid containing the genetic instructions used in the development and functioning of all known living organisms (with the exception of RNA viruses). The DNA segments carrying this genetic information are called genes. Likewise, other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information. Along with RNA and proteins, DNA is one of the three major macromolecules that are essential for all known forms of life. DNA consists of two long polymers of simple units called nucleotides, with backbones made of sugars and phosphate groups joined by ester bonds. These two strands run in opposite directions to each other and are therefore anti-parallel. Attached to each sugar is one of four types of molecules called nucleobases (informally, bases). It is the sequence of these four nucleobases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA in a process called transcription. Within cells DNA is organized into long structures called chromosomes. During cell division these chromosomes are duplicated in the process of DNA replication, providing each cell its own complete set of chromosomes. Eukaryotic organisms (animals, plants, fungi, and protists) store most of their DNA inside the cell nucleus and some of their DNA in organelles, such as mitochondria or chloroplasts.[1] In contrast, prokaryotes (bacteria and archaea) store their DNA only in the cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.

Ribonucleic acid Ribonucleic acid (RNA) functions in converting genetic information from genes into the amino acid sequences of proteins. The three universal types of RNA include transfer RNA (tRNA), messenger RNA (mRNA), and ribosomal RNA (rRNA). Messenger RNA acts to carry genetic sequence information between DNA and ribosomes, directing protein synthesis. Ribosomal RNA is a major component of the ribosome, and catalyzes peptide bond formation. Transfer RNA serves as the carrier molecule for amino acids to be used in protein synthesis, and is responsible for decoding the mRNA. In addition, many other classes of RNA are now known.

Artificial nucleic acid analogs Artificial nucleic acid analogs have been designed and synthesized by chemists, and include peptide nucleic acid, morpholino- and locked nucleic acid, as well as glycol nucleic acid and threose nucleic acid. Each of these is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule.

References [3] Mullis, Kary B. The Polymerase Chain Reaction (Nobel Lecture). 1993. (retrieved December 1, 2010) http:/ / nobelprize. org/ nobel_prizes/ chemistry/ laureates/ 1993/ mullis-lecture. html [9] Gilbert, Walter G. 1980. DNA Sequencing and Gene Structure (Nobel Lecture) http:/ / nobelprize. org/ nobel_prizes/ chemistry/ laureates/ 1980/ gilbert-lecture. html [10] Sanger, Frederick. 1980. Determination of Nucleotide Sequences in DNA (Nobel Lecture) http:/ / nobelprize. org/ nobel_prizes/ chemistry/ laureates/ 1980/ sanger-lecture. html

Nucleic acid

Further reading • Wolfram Saenger, Principles of Nucleic Acid Structure, 1984, Springer-Verlag New York Inc. • Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter Molecular Biology of the Cell, 2007, ISBN 978-0-8153-4105-5. Fourth edition is available online through the NCBI Bookshelf: link (http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4) • Jeremy M Berg, John L Tymoczko, and Lubert Stryer, Biochemistry 5th edition, 2002, W H Freeman. Available online through the NCBI Bookshelf: link (http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=stryer) • Astrid Sigel, Helmut Sigel and Roland K. O. Sigel, ed. (2012). Interplay between Metal Ions and Nucleic Acids. Metal Ions in Life Sciences 10. Springer. doi: 10.1007/978-94-007-2172-2 (http://dx.doi.org/10.1007/ 978-94-007-2172-2). ISBN978-94-007-2171-5.

External links • Interview with Aaron Klug, Nobel Laureate for structural elucidation of biologically important nucleic-acid protein complexes (http://www.vega.org.uk/video/programme/122) provided by the Vega Science Trust. • Nucleic Acids Research (Journal) (http://nar.oxfordjournals.org/) • Nucleic Acids Book (free online book on the chemistry and biology of nucleic acids) (http://www.atdbio.com/ nucleic-acids-book)

RNA

Ribonucleic acid (RNA) is a ubiquitous family of large biological molecules that perform multiple vital roles in the coding, decoding, regulation, and expression of genes. Together with DNA, RNA comprises the nucleic acids, which, along with proteins, constitute the three major macromolecules essential for all known forms of life. Like DNA, RNA is assembled as a chain of nucleotides, but is usually single-stranded. Cellular organisms use messenger RNA (mRNA) to convey genetic information (often notated using the letters G, A, U, and C for the nucleotides guanine, adenine, uracil and cytosine) that directs synthesis of specific proteins, while many viruses encode their genetic information using an RNA genome. Some RNA molecules play an active role within cells by catalyzing biological reactions, controlling gene expression, or sensing and communicating responses to cellular signals. One of these active processes is protein synthesis, a universal function whereby mRNA molecules direct the assembly of proteins on ribosomes. This process uses transfer RNA (tRNA) molecules to deliver amino acids to the ribosome, where ribosomal RNA (rRNA) links amino acids together to form proteins. A hairpin loop from a pre-mRNA. Highlighted are the nucleobases (green) and the ribose-phosphate backbone (blue). Note that this is a single strand of RNA that folds back upon itself.

Comparison with DNA The chemical structure of RNA is very similar to that of DNA, but differs in three main ways: • Unlike double-stranded DNA, RNA is a single-stranded molecule in many of its biological roles and has a much shorter chain of nucleotides. However, RNA can, by complementary base pairing, form intrastrand double helixes, as in tRNA. • While DNA contains deoxyribose, RNA contains ribose (in deoxyribose there is no hydroxyl group attached to the pentose ring in the 2' position). These hydroxyl groups make RNA less stable than DNA because it is more prone to hydrolysis. • The complementary base to adenine is not thymine, as it is in DNA, but rather uracil, which is an unmethylated form of thymine.[]

Three-dimensional representation of the 50S ribosomal subunit. RNA is in ochre, protein in blue. The active site is in the middle (red).

Like DNA, most biologically active RNAs, including mRNA, tRNA, rRNA, snRNAs, and other non-coding RNAs, contain self-complementary sequences that allow parts of the RNA to fold[1] and pair with itself to form double helices. Analysis of these RNAs has revealed that they are highly structured. Unlike DNA, their structures do not consist of

RNA

long double helices but rather collections of short helices packed together into structures akin to proteins. In this fashion, RNAs can achieve chemical catalysis, like enzymes.[2] For instance, determination of the structure of the ribosome—an enzyme that catalyzes peptide bond formation—revealed that its active site is composed entirely of RNA.[]

Structure Each nucleotide in RNA contains a ribose sugar, with carbons numbered 1' through 5'. A base is attached to the 1' position, in general, adenine (A), cytosine (C), guanine (G), or uracil (U). Adenine and guanine are purines, cytosine, and uracil are pyrimidines. A phosphate group is attached to the 3' position of one ribose and the 5' position of the next. The phosphate groups have a negative charge each at physiological pH, making RNA a charged molecule (polyanion). The bases may form hydrogen bonds between cytosine and guanine, between adenine and uracil and between guanine and uracil.[] However, other interactions are possible, such as a group of adenine bases binding to each other in a bulge,[3] or the GNRA tetraloop that has a guanine–adenine base-pair.[]

Watson-Crick base pairs in a siRNA (hydrogen atoms are not shown)

An important structural feature of RNA that distinguishes it from DNA is the presence of a hydroxyl group at the 2' position of the ribose sugar. The presence of this functional group causes the helix to adopt the A-form geometry rather than the B-form most commonly observed in DNA.[4] This results in a very deep and narrow major groove and a shallow and wide minor groove.[5] A second consequence of the presence of the 2'-hydroxyl group is that in conformationally flexible regions of an RNA molecule (that is, not involved in formation of a double helix), it can chemically attack the adjacent phosphodiester bond to cleave the backbone.[6] Chemical structure of RNA

RNA is transcribed with only four bases (adenine, cytosine, guanine and uracil),[7] but these bases and attached sugars can be modified in numerous ways as the RNAs mature. Pseudouridine (Ψ), in which the linkage between uracil and ribose is changed from a C–N bond to a C–C bond, and ribothymidine (T) are found in various places (the most notable ones being in the TΨC loop of tRNA).[8] Another notable modified base is hypoxanthine, a deaminated adenine base whose nucleoside is called inosine (I). Inosine plays a key role in the wobble hypothesis of the genetic code.[9]

Secondary structure of a telomerase RNA.

RNA There are nearly 100 other naturally occurring modified nucleosides,[10] of which pseudouridine and nucleosides with 2'-O-methylribose are the most common.[11] The specific roles of many of these modifications in RNA are not fully understood. However, it is notable that, in ribosomal RNA, many of the post-transcriptional modifications occur in highly functional regions, such as the peptidyl transferase center and the subunit interface, implying that they are important for normal function.[12] The functional form of single-stranded RNA molecules, just like proteins, frequently requires a specific tertiary structure. The scaffold for this structure is provided by secondary structural elements that are hydrogen bonds within the molecule. This leads to several recognizable "domains" of secondary structure like hairpin loops, bulges, and internal loops.[13] Since RNA is charged, metal ions such as Mg2+ are needed to stabilise many secondary and tertiary structures.[14]

Synthesis Synthesis of RNA is usually catalyzed by an enzyme—RNA polymerase—using DNA as a template, a process known as transcription. Initiation of transcription begins with the binding of the enzyme to a promoter sequence in the DNA (usually found "upstream" of a gene). The DNA double helix is unwound by the helicase activity of the enzyme. The enzyme then progresses along the template strand in the 3’ to 5’ direction, synthesizing a complementary RNA molecule with elongation occurring in the 5’ to 3’ direction. The DNA sequence also dictates where termination of RNA synthesis will occur.[15] RNAs are often modified by enzymes after transcription. For example, a poly(A) tail and a 5' cap are added to eukaryotic pre-mRNA and introns are removed by the spliceosome. There are also a number of RNA-dependent RNA polymerases that use RNA as their template for synthesis of a new strand of RNA. For instance, a number of RNA viruses (such as poliovirus) use this type of enzyme to replicate their genetic material.[16] Also, RNA-dependent RNA polymerase is part of the RNA interference pathway in many organisms.[17]

RNA

Types of RNA Overview Messenger RNA (mRNA) is the RNA that carries information from DNA to the ribosome, the sites of protein synthesis (translation) in the cell. The coding sequence of the mRNA determines the amino acid sequence in the protein that is produced.[] Many RNAs do not code for protein however (about 97% of the transcriptional output is non-protein-coding in eukaryotes [18][19][20][21]). These so-called non-coding RNAs ("ncRNA") can be encoded by their own genes (RNA genes), but can also derive from mRNA introns.[] The most prominent examples of non-coding RNAs are transfer RNA (tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation.[] There are also non-coding RNAs involved in gene regulation, RNA processing and other roles. Certain RNAs are able to catalyse chemical reactions such as cutting and ligating other RNA molecules,[22] and the catalysis of peptide bond formation in the ribosome;[] these are known as ribozymes.

In translation Messenger RNA (mRNA) carries information about a protein sequence Structure of a hammerhead ribozyme, a ribozyme to the ribosomes, the protein synthesis factories in the cell. It is coded that cuts RNA so that every three nucleotides (a codon) correspond to one amino acid. In eukaryotic cells, once precursor mRNA (pre-mRNA) has been transcribed from DNA, it is processed to mature mRNA. This removes its introns—non-coding sections of the pre-mRNA. The mRNA is then exported from the nucleus to the cytoplasm, where it is bound to ribosomes and translated into its corresponding protein form with the help of tRNA. In prokaryotic cells, which do not have nucleus and cytoplasm compartments, mRNA can bind to ribosomes while it is being transcribed from DNA. After a certain amount of time the message degrades into its component nucleotides with the assistance of ribonucleases.[] Transfer RNA (tRNA) is a small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites for amino acid attachment and an anticodon region for codon recognition that binds to a specific sequence on the messenger RNA chain through hydrogen bonding.[] Ribosomal RNA (rRNA) is the catalytic component of the ribosomes. Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA. Three of the rRNA molecules are synthesized in the nucleolus, and one is synthesized elsewhere. In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a ribosome. The ribosome binds mRNA and carries out protein synthesis. Several ribosomes may be attached to a single mRNA at any time.[] Nearly all the RNA found in a typical eukaryotic cell is rRNA. Transfer-messenger RNA (tmRNA) is found in many bacteria and plastids. It tags proteins encoded by mRNAs that lack stop codons for degradation and prevents the ribosome from stalling.[23]

RNA

Regulatory RNAs Several types of RNA can downregulate gene expression by being complementary to a part of an mRNA or a gene's DNA. MicroRNAs (miRNA; 21-22nt) are found in eukaryotes and act through RNA interference (RNAi), where an effector complex of miRNA and enzymes can cleave complementary mRNA, block the mRNA from being translated, or accelerate its degradation.[24][25] While small interfering RNAs (siRNA; 20-25nt) are often produced by breakdown of viral RNA, there are also endogenous sources of siRNAs.[26][27] siRNAs act through RNA interference in a fashion similar to miRNAs. Some miRNAs and siRNAs can cause genes they target to be methylated, thereby decreasing or increasing transcription of those genes.[28][29][30] Animals have Piwi-interacting RNAs (piRNA; 29-30nt) that are active in germline cells and are thought to be a defense against transposons and play a role in gametogenesis.[][31] Many prokaryotes have CRISPR RNAs, a regulatory system similar to RNA interference.[32] Antisense RNAs are widespread; most downregulate a gene, but a few are activators of transcription.[33] One way antisense RNA can act is by binding to an mRNA, forming double-stranded RNA that is enzymatically degraded.[34] There are many long noncoding RNAs that regulate genes in eukaryotes,[35] one such RNA is Xist, which coats one X chromosome in female mammals and inactivates it.[36] An mRNA may contain regulatory elements itself, such as riboswitches, in the 5' untranslated region or 3' untranslated region; these cis-regulatory elements regulate the activity of that mRNA.[37] The untranslated regions can also contain elements that regulate other genes.[38]

In RNA processing Many RNAs are involved in modifying other RNAs. Introns are spliced out of pre-mRNA by spliceosomes, which contain several small nuclear RNAs (snRNA),[] or the introns can be ribozymes that are spliced by themselves.[39] RNA can also be altered by having its nucleotides modified to other nucleotides than A, C, G and U. In eukaryotes, modifications of RNA nucleotides are in general directed by small nucleolar RNAs (snoRNA; 60-300nt),[] found in the Uridine to pseudouridine is a common RNA modification. nucleolus and cajal bodies. snoRNAs associate with enzymes and guide them to a spot on an RNA by basepairing to that RNA. These enzymes then perform the nucleotide modification. rRNAs and tRNAs are extensively modified, but snRNAs and mRNAs can also be the target of base modification.[40][41] RNA can also be methylated.[42][43]

RNA genomes Like DNA, RNA can carry genetic information. RNA viruses have genomes composed of RNA that encodes a number of proteins. The viral genome is replicated by some of those proteins, while other proteins protect the genome as the virus particle moves to a new host cell. Viroids are another group of pathogens, but they consist only of RNA, do not encode any protein and are replicated by a host plant cell's polymerase.[44]

In reverse transcription Reverse transcribing viruses replicate their genomes by reverse transcribing DNA copies from their RNA; these DNA copies are then transcribed to new RNA. Retrotransposons also spread by copying DNA and RNA from one another,[45] and telomerase contains an RNA that is used as template for building the ends of eukaryotic chromosomes.[46]

RNA

Double-stranded RNA Double-stranded RNA (dsRNA) is RNA with two complementary strands, similar to the DNA found in all cells. dsRNA forms the genetic material of some viruses (double-stranded RNA viruses). Double-stranded RNA such as viral RNA or siRNA can trigger RNA interference in eukaryotes, as well as interferon response in vertebrates.[47][48][49][50]

Key discoveries in RNA biology Research on RNA has led to many important biological discoveries and numerous Nobel Prizes. Nucleic acids were discovered in 1868 by Friedrich Miescher, who called the material 'nuclein' since it was found in the nucleus.[51] It was later discovered that prokaryotic cells, which do not have a nucleus, also contain nucleic acids. The role of RNA in protein synthesis was suspected already in 1939.[52] Severo Ochoa won the 1959 Nobel Prize in Medicine (shared with Arthur Kornberg) after he discovered an enzyme that can synthesize RNA in the laboratory.[53] However, the enzyme discovered by Ochoa (polynucleotide phosphorylase) was later shown to be responsible for RNA degradation, not RNA synthesis.

Robert W. Holley, left, poses with his research team.

The sequence of the 77 nucleotides of a yeast tRNA was found by Robert W. Holley in 1965,[54] winning Holley the 1968 Nobel Prize in Medicine (shared with Har Gobind Khorana and Marshall Nirenberg). In 1967, Carl Woese hypothesized that RNA might be catalytic and suggested that the earliest forms of life (self-replicating molecules) could have relied on RNA both to carry genetic information and to catalyze biochemical reactions—an RNA world.[55][56] During the early 1970s retroviruses and reverse transcriptase were discovered, showing for the first time that enzymes could copy RNA into DNA (the opposite of the usual route for transmission of genetic information). For this work, David Baltimore, Renato Dulbecco and Howard Temin were awarded a Nobel Prize in 1975. In 1976, Walter Fiers and his team determined the first complete nucleotide sequence of an RNA virus genome, that of bacteriophage MS2.[57] In 1977, introns and RNA splicing were discovered in both mammalian viruses and in cellular genes, resulting in a 1993 Nobel to Philip Sharp and Richard Roberts. Catalytic RNA molecules (ribozymes) were discovered in the early 1980s, leading to a 1989 Nobel award to Thomas Cech and Sidney Altman. In 1990 it was found in petunia that introduced genes can silence similar genes of the plant's own, now known to be a result of RNA interference.[58][59] At about the same time, 22 nt long RNAs, now called microRNAs, were found to have a role in the development of C. elegans.[60] Studies on RNA interference gleaned a Nobel Prize for Andrew Fire and Craig Mello in 2006, and another Nobel was awarded for studies on transcription of RNA to Roger Kornberg in the same year. The discovery of gene regulatory RNAs has led to attempts to develop drugs made of RNA, such as siRNA, to silence genes.[61]

RNA

References [1] Papercore summary (http:/ / papercore. org/ Tinoco1999)

External links • RNA World website (http://www.imb-jena.de/RNA.html) Link collection (structures, sequences, tools, journals) • Nucleic Acid Database (http://ndbserver.rutgers.edu/atlas/xray/) Images of DNA, RNA and complexes. • EteRNA (http://eterna.cmu.edu/content/EteRNA) a game forming RNA by pairing bases.

DNA Deoxyribonucleic acid (DNA) is a molecule that encodes the genetic instructions used in the development and functioning of all known living organisms and many viruses. Along with RNA and proteins, DNA is one of the three major macromolecules essential for all known forms of life. Genetic information is encoded as a sequence of nucleotides (guanine, adenine, thymine, and cytosine) recorded using the letters G, A, T, and C. Most DNA molecules are double-stranded helices, consisting of two long polymers of simple units called nucleotides, molecules with backbones made of alternating sugars (deoxyribose) and phosphate groups (related to phosphoric acid), with the nucleobases (G, A, T, C) attached to The structure of the DNA double helix. The atoms in the structure are colour-coded by the sugars. DNA is well-suited for element and the detailed structure of two base pairs are shown in the bottom right. biological information storage, since the DNA backbone is resistant to cleavage and the double-stranded structure provides the molecule with a built-in duplicate of the encoded information. These two strands run in opposite directions to each other and are therefore anti-parallel, one backbone being 3′ (three prime) and the other 5′

DNA

(five prime). This refers to the direction the 3rd and 5th carbon on the sugar molecule is facing. Attached to each sugar is one of four types of molecules called nucleobases (informally, bases). It is the sequence of these four nucleobases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA in a process called transcription. Within cells, DNA is organized into long structures called chromosomes. During cell division these chromosomes are duplicated in the process of DNA replication, providing each cell its own complete set of chromosomes. Eukaryotic organisms (animals, plants, fungi, and protists) store most of their DNA inside the cell nucleus and some of their DNA in organelles, such as mitochondria or chloroplasts.[1] In contrast, prokaryotes (bacteria and archaea) store their DNA only in the cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed. The structure of part of a DNA double helix

DNA

Properties DNA is a long polymer made from repeating units called nucleotides.[2][][3] DNA was first identified and isolated by Friedrich Miescher and the double helix structure of DNA was first discovered by James Watson and Francis Crick. The structure of DNA of all species comprises two helical chains each coiled round the same axis, and each with a pitch of 34ångströms (3.4nanometres) and a radius of 10ångströms (1.0nanometres).[] According to another study, when measured in a particular solution, the DNA chain measured 22 to 26ångströms wide (2.2 to 2.6nanometres), and one nucleotide unit measured 3.3Å (0.33nm) long.[4] Although each individual repeating unit is very small, DNA polymers can be very large molecules containing millions of nucleotides. For instance, the largest human chromosome, chromosome number 1, consists of approximately 220 million base pairs[5] and is 85 mm long.

Chemical structure of DNA. Hydrogen bonds shown as dotted lines.

In living organisms DNA does not usually exist as a single molecule, but instead as a pair of molecules that are held tightly together.[][6] These two long strands entwine like vines, in the shape of a double helix. The nucleotide repeats contain both the segment of the backbone of the molecule, which holds the chain together, and a nucleobase, which interacts with the other DNA strand in the helix. A nucleobase linked to a sugar is called a nucleoside and a base linked to a sugar and one or more phosphate groups is called a nucleotide. A polymer comprising multiple linked nucleotides (as in DNA) is called a polynucleotide.[7] The backbone of the DNA strand is made from alternating phosphate and sugar residues.[] The sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds mean a strand of DNA has a direction. In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel. The asymmetric ends of DNA strands are called the 5′ (five prime) and 3′ (three prime) ends, with the 5′ end having a terminal phosphate group and the 3′ end a terminal hydroxyl group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA.[6]

DNA

The DNA double helix is stabilized primarily by two forces: hydrogen bonds between nucleotides and base-stacking interactions among aromatic nucleobases.[] In the aqueous environment of the cell, the conjugated π bonds of nucleotide bases align perpendicular to the axis of the DNA molecule, minimizing their interaction with the solvation shell and therefore, the Gibbs free energy. The four bases found in DNA are adenine (abbreviated A), cytosine (C), guanine (G) and thymine (T). These four bases are attached to the sugar/phosphate to form the complete nucleotide, as shown for adenosine monophosphate.

Nucleobase classification The nucleobases are classified into two types: the purines, A and G, being fused five- and six-membered heterocyclic compounds, and the pyrimidines, the six-membered rings C and T.[6] A fifth pyrimidine nucleobase, uracil (U), usually takes the place of thymine in RNA and differs from thymine by lacking a methyl group on its ring. In addition to RNA and DNA a large number of artificial nucleic acid analogues have also been created to study the properties of nucleic acids, or for use in biotechnology.[9]

A section of DNA. The bases lie horizontally between the two [8] spiraling strands. (animated version).

Uracil is not usually found in DNA, occurring only as a breakdown product of cytosine. However in a number of bacteriophages – Bacillus subtilis bacteriophages PBS1 and PBS2 and Yersinia bacteriophage piR1-37 – thymine has been replaced by uracil.[] Base J (beta-d-glucopyranosyloxymethyluracil), a modified form of uracil, is also found in a number of organisms: the flagellates Diplonema and Euglena, and all the kinetoplastid genera[] Biosynthesis of J occurs in two steps: in the first step a specific thymidine in DNA is converted into hydroxymethyldeoxyuridine; in the second HOMedU is glycosylated to form J.[] Proteins that bind specifically to this base have been identified.[][][] These proteins appear to be distant relatives of the Tet1 oncogene that is involved in the pathogenesis of acute myeloid leukemia.[] J appears to act as a termination signal for RNA polymerase II.[][]

Grooves Twin helical strands form the DNA backbone. Another double helix may be found tracing the spaces, or grooves, between the strands. These voids are adjacent to the base pairs and may provide a binding site. As the strands are not symmetrically located with respect to each other, the grooves are unequally sized. One groove, the major groove, is 22Å wide and the other, the minor groove, is 12Å wide.[10] The narrowness of the minor groove means that the edges of the bases are Major and minor grooves of DNA. Minor groove more accessible in the major groove. As a result, proteins like is a binding site for the dye Hoechst 33258. transcription factors that can bind to specific sequences in double-stranded DNA usually make contacts to the sides of the bases exposed in the major groove.[] This situation varies in unusual conformations of DNA within the cell (see below), but the major and minor grooves are always named to reflect the differences in size that would be seen if the DNA is twisted back into the ordinary B form.

DNA

Base pairing In a DNA double helix, each type of nucleobase on one strand bonds with just one type of nucleobase on the other strand. This is called complementary base pairing. Here, purines form hydrogen bonds to pyrimidines, with adenine bonding only to thymine in two hydrogen bonds, and cytosine bonding only to guanine in three hydrogen bonds. This arrangement of two nucleotides binding together across the double helix is called a base pair. As hydrogen bonds are not covalent, they can be broken and rejoined relatively easily. The two strands of DNA in a double helix can therefore be pulled apart like a zipper, either by a mechanical force or high temperature.[11] As a result of this complementarity, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA replication. Indeed, this reversible and specific interaction between complementary base pairs is critical for all the functions of DNA in living organisms.[]

Top, a GC base pair with three hydrogen bonds. Bottom, an AT base pair with two hydrogen bonds. Non-covalent hydrogen bonds between the pairs are shown as dashed lines. The two types of base pairs form different numbers of hydrogen bonds, AT forming two hydrogen bonds, and GC forming three hydrogen bonds (see figures, right). DNA with high GC-content is more stable than DNA with low GC-content. As noted above, most DNA molecules are actually two polymer strands, bound together in a helical fashion by noncovalent bonds; this double stranded structure (dsDNA) is maintained largely by the intrastrand base stacking interactions, which are strongest for G,C stacks. The two strands can come apart– a process known as melting– to form two ssDNA molecules. Melting occurs when conditions favor ssDNA; such conditions are high temperature, low salt and high pH (low pH also melts DNA, but since DNA is unstable due to acid depurination, low pH is rarely used). The stability of the dsDNA form depends not only on the GC-content (% G,C basepairs) but also on sequence (since stacking is sequence specific) and also length (longer molecules are more stable). The stability can be measured in various ways; a common way is the "melting temperature", which is the temperature at which 50% of the ds molecules are converted to ss molecules; melting temperature is dependent on ionic strength and the concentration of DNA. As a result, it is both the percentage of GC base pairs and the overall length of a DNA double helix that determines the strength of the association between the two strands of DNA. Long DNA helices with a high GC-content have stronger-interacting strands, while short helices with high AT content have weaker-interacting strands.[12] In biology, parts of the DNA double helix that need to separate easily, such as the TATAAT Pribnow box in some promoters, tend to have a high AT content, making the strands easier to pull apart.[13] In the laboratory, the strength of this interaction can be measured by finding the temperature necessary to break the hydrogen bonds, their melting temperature (also called Tm value). When all the base pairs in a DNA double helix melt, the strands separate and exist in solution as two entirely independent molecules. These single-stranded DNA molecules (ssDNA) have no single common shape, but some conformations are more stable than others.[14]

DNA

Sense and antisense A DNA sequence is called "sense" if its sequence is the same as that of a messenger RNA copy that is translated into protein.[15] The sequence on the opposite strand is called the "antisense" sequence. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands contain both sense and antisense sequences). In both prokaryotes and eukaryotes, antisense RNA sequences are produced, but the functions of these RNAs are not entirely clear.[16] One proposal is that antisense RNAs are involved in regulating gene expression through RNA-RNA base pairing.[17] A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses, blur the distinction between sense and antisense strands by having overlapping genes.[18] In these cases, some DNA sequences do double duty, encoding one protein when read along one strand, and a second protein when read in the opposite direction along the other strand. In bacteria, this overlap may be involved in the regulation of gene transcription,[19] while in viruses, overlapping genes increase the amount of information that can be encoded within the small viral genome.[20]

Supercoiling DNA can be twisted like a rope in a process called DNA supercoiling. With DNA in its "relaxed" state, a strand usually circles the axis of the double helix once every 10.4 base pairs, but if the DNA is twisted the strands become more tightly or more loosely wound.[21] If the DNA is twisted in the direction of the helix, this is positive supercoiling, and the bases are held more tightly together. If they are twisted in the opposite direction, this is negative supercoiling, and the bases come apart more easily. In nature, most DNA has slight negative supercoiling that is introduced by enzymes called topoisomerases.[] These enzymes are also needed to relieve the twisting stresses introduced into DNA strands during processes such as transcription and DNA replication.[]

Alternate DNA structures DNA exists in many possible conformations that include A-DNA, B-DNA, and Z-DNA forms, although, only B-DNA and Z-DNA have been directly observed in functional organisms.[] The conformation that DNA adopts depends on the hydration level, DNA sequence, the amount and direction of supercoiling, chemical modifications of the bases, the type and concentration of metal ions, as well as the presence of polyamines in solution.[22]

From left to right, the structures of A, B and Z DNA

The first published reports of A-DNA X-ray diffraction patterns— and also B-DNA — used analyses based on Patterson transforms that provided only a limited amount of structural information for oriented fibers of DNA.[23][] An alternate analysis was then proposed by Wilkins et al., in 1953, for the in vivo B-DNA X-ray diffraction/scattering patterns of highly hydrated DNA fibers in terms of squares of Bessel functions.[] In the same journal, James Watson and Francis Crick presented their molecular modeling analysis of the DNA X-ray diffraction patterns to suggest that the structure was a double-helix.[] Although the `B-DNA form' is most common under the conditions found in cells,[24] it is not a well-defined conformation but a family of related DNA conformations[25] that occur at the high hydration levels present in living cells. Their corresponding X-ray diffraction and scattering patterns are characteristic of molecular paracrystals with a significant degree of disorder.[26][27] Compared to B-DNA, the A-DNA form is a wider right-handed spiral, with a shallow, wide minor groove and a narrower, deeper major groove. The A form occurs under non-physiological conditions in partially dehydrated samples of DNA, while in the cell it may be produced in hybrid pairings of DNA and RNA strands, as well as in enzyme-DNA complexes.[28][29] Segments of DNA where the bases have been chemically modified by methylation

DNA

may undergo a larger change in conformation and adopt the Z form. Here, the strands turn about the helical axis in a left-handed spiral, the opposite of the more common B form.[30] These unusual structures can be recognized by specific Z-DNA binding proteins and may be involved in the regulation of transcription.[31]

Alternate DNA chemistry For a number of years exobiologists have proposed the existence of a shadow biosphere, a postulated microbial biosphere of Earth that uses radically different biochemical and molecular processes than currently known life. One of the proposals was the existence of lifeforms that use arsenic instead of phosphorus in DNA. A report in 2010 of the possibility in the bacterium GFAJ-1, was announced,[][][] though the research was disputed,[][32] and evidence suggests the bacterium actively prevents the incorporation of arsenic into the DNA backbone and other biomolecules.[]

Quadruplex structures At the ends of the linear chromosomes are specialized regions of DNA called telomeres. The main function of these regions is to allow the cell to replicate chromosome ends using the enzyme telomerase, as the enzymes that normally replicate DNA cannot copy the extreme 3′ ends of chromosomes.[] These specialized chromosome caps also help protect the DNA ends, and stop the DNA repair systems in the cell from treating them as damage to be corrected.[] In human cells, telomeres are usually lengths of single-stranded DNA containing several thousand repeats of a simple TTAGGG sequence.[33] These guanine-rich sequences may stabilize chromosome ends by forming structures of stacked sets of four-base units, rather than the usual base pairs found in other DNA molecules. Here, four guanine bases form a flat plate and these flat four-base units then stack on top of each other, to form a stable G-quadruplex structure.[] These structures are stabilized by hydrogen bonding between the edges of the bases and chelation of a metal ion in the centre of each four-base unit.[35] Other structures can also be formed, with the central set of four bases coming from either a single strand folded around the bases, or several different parallel strands, each contributing one base to the central structure.

DNA quadruplex formed by telomere repeats. The looped conformation of the DNA backbone [34] is very different from the typical DNA helix.

In addition to these stacked structures, telomeres also form large loop structures called telomere loops, or T-loops. Here, the single-stranded DNA curls around in a long circle stabilized by telomere-binding proteins.[36] At the very end of the T-loop, the single-stranded telomere DNA is held onto a region of double-stranded DNA by the telomere strand disrupting the double-helical DNA and base pairing to one of the two strands. This triple-stranded structure is called a displacement loop or D-loop.[]

Single branch

Branched DNA can form networks containing multiple branches.

Multiple branches

DNA

Branched DNA In DNA fraying occurs when non-complementary regions exist at the end of an otherwise complementary double-strand of DNA. However, branched DNA can occur if a third strand of DNA is introduced and contains adjoining regions able to hybridize with the frayed regions of the pre-existing double-strand. Although the simplest example of branched DNA involves only three strands of DNA, complexes involving additional strands and multiple branches are also possible.[37] Branched DNA can be used in nanotechnology to construct geometric shapes, see the section on uses in technology below.

Vibration DNA may carry out low-frequency collective motion as observed by the Raman spectroscopy[][] and analyzed with a quasi-continuum model.[][]

Chemical modifications and altered DNA packaging

cytosine

5-methylcytosine

thymine

Structure of cytosine with and without the 5-methyl group. Deamination converts 5-methylcytosine into thymine.

Base modifications and DNA packaging The expression of genes is influenced by how the DNA is packaged in chromosomes, in a structure called chromatin. Base modifications can be involved in packaging, with regions that have low or no gene expression usually containing high levels of methylation of cytosine bases. DNA packaging and its influence on gene expression can also occur by covalent modifications of the histone protein core around which DNA is wrapped in the chromatin structure or else by remodeling carried out by chromatin remodeling complexes (see Chromatin remodeling). There is, further, crosstalk between DNA methylation and histone modification, so they can coordinately affect chromatin and gene expression.[38] For one example, cytosine methylation, produces 5-methylcytosine, which is important for X-chromosome inactivation.[39] The average level of methylation varies between organisms– the worm Caenorhabditis elegans lacks cytosine methylation, while vertebrates have higher levels, with up to 1% of their DNA containing 5-methylcytosine.[40] Despite the importance of 5-methylcytosine, it can deaminate to leave a thymine base, so methylated cytosines are particularly prone to mutations.[41] Other base modifications include adenine methylation in bacteria, the presence of 5-hydroxymethylcytosine in the brain,[42] and the glycosylation of uracil to produce the "J-base" in kinetoplastids.[43][44]

DNA

Damage DNA can be damaged by many sorts of mutagens, which change the DNA sequence. Mutagens include oxidizing agents, alkylating agents and also high-energy electromagnetic radiation such as ultraviolet light and X-rays. The type of DNA damage produced depends on the type of mutagen. For example, UV light can damage DNA by producing thymine dimers, which are cross-links between pyrimidine bases.[46] On the other hand, oxidants such as free radicals or hydrogen peroxide produce multiple forms of damage, including base modifications, particularly of guanosine, and double-strand breaks.[47] A typical human cell contains about 150,000 bases that have suffered oxidative damage.[48] Of these oxidative lesions, the most dangerous are double-strand breaks, as these are difficult to repair and can produce point mutations, insertions and deletions from the DNA sequence, as well as chromosomal translocations.[49] These mutations can cause cancer. Because of inherent limitations in the DNA repair mechanisms, if humans lived long enough, they would all eventually develop A covalent adduct between a metabolically cancer.[][50] DNA damages that are naturally occurring, due to normal activated form of benzo[a]pyrene, the major [45] cellular processes that produce reactive oxygen species, the hydrolytic mutagen in tobacco smoke, and DNA activities of cellular water, etc., also occur frequently. Although most of these damages are repaired, in any cell some DNA damage may remain despite the action of repair processes. These remaining DNA damages accumulate with age in mammalian postmitotic tissues. This accumulation appears to be an important underlying cause of aging.[51][52][53] Many mutagens fit into the space between two adjacent base pairs, this is called intercalation. Most intercalators are aromatic and planar molecules; examples include ethidium bromide, acridines, daunomycin, and doxorubicin. For an intercalator to fit between base pairs, the bases must separate, distorting the DNA strands by unwinding of the double helix. This inhibits both transcription and DNA replication, causing toxicity and mutations.[54] As a result, DNA intercalators may be carcinogens, and in the case of thalidomide, a teratogen.[55] Others such as benzo[a]pyrene diol epoxide and aflatoxin form DNA adducts that induce errors in replication.[56] Nevertheless, due to their ability to inhibit DNA transcription and replication, other similar toxins are also used in chemotherapy to inhibit rapidly growing cancer cells.[57]

Biological functions DNA usually occurs as linear chromosomes in eukaryotes, and circular chromosomes in prokaryotes. The set of chromosomes in a cell makes up its genome; the human genome has approximately 3 billion base pairs of DNA arranged into 46 chromosomes.[58] The information carried by DNA is held in the sequence of pieces of DNA called genes. Transmission of genetic information in genes is achieved via complementary base pairing. For example, in transcription, when a cell uses the information in a gene, the DNA sequence is copied into a complementary RNA sequence through the attraction between the DNA and the correct RNA nucleotides. Usually, this RNA copy is then used to make a matching protein sequence in a process called translation, which depends on the same interaction between RNA nucleotides. In alternative fashion, a cell may simply copy its genetic information in a process called DNA replication. The details of these functions are covered in other articles; here we focus on the interactions between DNA and other molecules that mediate the function of the genome.

DNA

Genes and genomes Genomic DNA is tightly and orderly packed in the process called DNA condensation to fit the small available volumes of the cell. In eukaryotes, DNA is located in the cell nucleus, as well as small amounts in mitochondria and chloroplasts. In prokaryotes, the DNA is held within an irregularly shaped body in the cytoplasm called the nucleoid.[59] The genetic information in a genome is held within genes, and the complete set of this information in an organism is called its genotype. A gene is a unit of heredity and is a region of DNA that influences a particular characteristic in an organism. Genes contain an open reading frame that can be transcribed, as well as regulatory sequences such as promoters and enhancers, which control the transcription of the open reading frame. In many species, only a small fraction of the total sequence of the genome encodes protein. For example, only about 1.5% of the human genome consists of protein-coding exons, with over 50% of human DNA consisting of non-coding repetitive sequences.[60] The reasons for the presence of so much noncoding DNA in eukaryotic genomes and the extraordinary differences in genome size, or C-value, among species represent a long-standing puzzle known as the "C-value enigma".[61] However, some DNA sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation of gene expression.[62] Some noncoding DNA sequences play structural roles in chromosomes. Telomeres and centromeres typically contain few genes, but are important for the function and stability of chromosomes.[][64] An abundant form of noncoding DNA in humans are pseudogenes, which are copies of genes that have been disabled by mutation.[65] These sequences are usually just molecular fossils, although they can occasionally serve as raw genetic material for the creation of new genes through the process of gene duplication and divergence.[66]

Transcription and translation

T7 RNA polymerase (blue) producing a mRNA [63] (green) from a DNA template (orange).

A gene is a sequence of DNA that contains genetic information and can influence the phenotype of an organism. Within a gene, the sequence of bases along a DNA strand defines a messenger RNA sequence, which then defines one or more protein sequences. The relationship between the nucleotide sequences of genes and the amino-acid sequences of proteins is determined by the rules of translation, known collectively as the genetic code. The genetic code consists of three-letter 'words' called codons formed from a sequence of three nucleotides (e.g. ACT, CAG, TTT). In transcription, the codons of a gene are copied into messenger RNA by RNA polymerase. This RNA copy is then decoded by a ribosome that reads the RNA sequence by base-pairing the messenger RNA to transfer RNA, which carries amino acids. Since there are 4 bases in 3-letter combinations, there are 64 possible codons ( combinations). These encode the twenty standard amino acids, giving most amino acids more than one possible codon. There are also three 'stop' or 'nonsense' codons signifying the end of the coding region; these are the TAA, TGA and TAG codons.

DNA

Replication Cell division is essential for an organism to grow, but, when a cell divides, it must replicate the DNA in its genome so that the two daughter cells have the same genetic information as their parent. The double-stranded structure of DNA provides a simple mechanism for DNA replication. Here, the two strands are DNA replication. The double helix is unwound by a helicase and topoisomerase. Next, separated and then each strand's one DNA polymerase produces the leading strand copy. Another DNA polymerase binds complementary DNA sequence is to the lagging strand. This enzyme makes discontinuous segments (called Okazaki fragments) before DNA ligase joins them together. recreated by an enzyme called DNA polymerase. This enzyme makes the complementary strand by finding the correct base through complementary base pairing, and bonding it onto the original strand. As DNA polymerases can only extend a DNA strand in a 5′ to 3′ direction, different mechanisms are used to copy the antiparallel strands of the double helix.[67] In this way, the base on the old strand dictates which base appears on the new strand, and the cell ends up with a perfect copy of its DNA.

Interactions with proteins All the functions of DNA depend on interactions with proteins. These protein interactions can be non-specific, or the protein can bind specifically to a single DNA sequence. Enzymes can also bind to DNA and of these, the polymerases that copy the DNA base sequence in transcription and DNA replication are particularly important.

DNA-binding proteins

Interaction of DNA (shown in orange) with histones (shown in blue). These proteins' basic amino acids bind to the acidic phosphate groups on DNA. Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin. In eukaryotes this structure involves DNA binding to a complex of small basic proteins called histones, while in prokaryotes multiple types of proteins are involved.[68][69] The histones form a disk-shaped complex called a nucleosome, which contains two complete turns of double-stranded DNA wrapped around its surface. These non-specific interactions are formed through basic residues in the histones making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are therefore largely independent of the base sequence.[70] Chemical modifications of these basic amino acid residues include methylation, phosphorylation and acetylation.[71] These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to transcription factors and changing the rate of transcription.[72] Other non-specific

DNA

DNA-binding proteins in chromatin include the high-mobility group proteins, which bind to bent or distorted DNA.[73] These proteins are important in bending arrays of nucleosomes and arranging them into the larger structures that make up chromosomes.[74] A distinct group of DNA-binding proteins are the DNA-binding proteins that specifically bind single-stranded DNA. In humans, replication protein A is the best-understood member of this family and is used in processes where the double helix is separated, including DNA replication, recombination and DNA repair.[75] These binding proteins seem to stabilize single-stranded DNA and protect it from forming stem-loops or being degraded by nucleases. In contrast, other proteins have evolved to bind to particular DNA sequences. The most intensively studied of these are the various transcription factors, which are proteins that regulate transcription. Each transcription factor binds to one particular set of DNA sequences and activates or inhibits the transcription of genes that have these sequences close to their promoters. The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription, either directly or through other mediator proteins; this locates the polymerase at the promoter and allows it to begin transcription.[77] Alternatively, transcription factors can bind enzymes that modify the histones at the promoter. This changes the accessibility of the DNA template to the polymerase.[78] As these DNA targets can occur throughout an organism's genome, changes in The lambda repressor the activity of one type of transcription factor can affect thousands of genes.[79] helix-turn-helix transcription factor Consequently, these proteins are often the targets of the signal transduction [76] bound to its DNA target processes that control responses to environmental changes or cellular differentiation and development. The specificity of these transcription factors' interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases, allowing them to "read" the DNA sequence. Most of these base-interactions are made in the major groove, where the bases are most accessible.[]

DNA-modifying enzymes Nucleases and ligases Nucleases are enzymes that cut DNA strands by catalyzing the hydrolysis of the phosphodiester bonds. Nucleases that hydrolyse nucleotides from the ends of DNA strands are called exonucleases, while endonucleases cut within strands. The most frequently used nucleases in molecular biology are the restriction endonucleases, which The restriction enzyme EcoRV (green) in a [80] cut DNA at specific sequences. For instance, the EcoRV enzyme complex with its substrate DNA shown to the left recognizes the 6-base sequence 5′-GATATC-3′ and makes a cut at the vertical line. In nature, these enzymes protect bacteria against phage infection by digesting the phage DNA when it enters the bacterial cell, acting as part of the restriction modification system.[81] In technology, these sequence-specific nucleases are used in molecular cloning and DNA fingerprinting. Enzymes called DNA ligases can rejoin cut or broken DNA strands.[] Ligases are particularly important in lagging strand DNA replication, as they join together the short segments of DNA produced at the replication fork into a complete copy of the DNA template. They are also used in DNA repair and genetic recombination.[]

DNA Topoisomerases and helicases Topoisomerases are enzymes with both nuclease and ligase activity. These proteins change the amount of supercoiling in DNA. Some of these enzymes work by cutting the DNA helix and allowing one section to rotate, thereby reducing its level of supercoiling; the enzyme then seals the DNA break.[] Other types of these enzymes are capable of cutting one DNA helix and then passing a second strand of DNA through this break, before rejoining the helix.[82] Topoisomerases are required for many processes involving DNA, such as DNA replication and transcription.[] Helicases are proteins that are a type of molecular motor. They use the chemical energy in nucleoside triphosphates, predominantly ATP, to break hydrogen bonds between bases and unwind the DNA double helix into single strands.[83] These enzymes are essential for most processes where enzymes need to access the DNA bases. Polymerases Polymerases are enzymes that synthesize polynucleotide chains from nucleoside triphosphates. The sequence of their products are copies of existing polynucleotide chains—which are called templates. These enzymes function by adding nucleotides onto the 3′ hydroxyl group of the previous nucleotide in a DNA strand. As a consequence, all polymerases work in a 5′ to 3′ direction.[] In the active site of these enzymes, the incoming nucleoside triphosphate base-pairs to the template: this allows polymerases to accurately synthesize the complementary strand of their template. Polymerases are classified according to the type of template that they use. In DNA replication, a DNA-dependent DNA polymerase makes a copy of a DNA sequence. Accuracy is vital in this process, so many of these polymerases have a proofreading activity. Here, the polymerase recognizes the occasional mistakes in the synthesis reaction by the lack of base pairing between the mismatched nucleotides. If a mismatch is detected, a 3′ to 5′ exonuclease activity is activated and the incorrect base removed.[84] In most organisms, DNA polymerases function in a large complex called the replisome that contains multiple accessory subunits, such as the DNA clamp or helicases.[85] RNA-dependent DNA polymerases are a specialized class of polymerases that copy the sequence of an RNA strand into DNA. They include reverse transcriptase, which is a viral enzyme involved in the infection of cells by retroviruses, and telomerase, which is required for the replication of telomeres.[][86] Telomerase is an unusual polymerase because it contains its own RNA template as part of its structure.[] Transcription is carried out by a DNA-dependent RNA polymerase that copies the sequence of a DNA strand into RNA. To begin transcribing a gene, the RNA polymerase binds to a sequence of DNA called a promoter and separates the DNA strands. It then copies the gene sequence into a messenger RNA transcript until it reaches a region of DNA called the terminator, where it halts and detaches from the DNA. As with human DNA-dependent DNA polymerases, RNA polymerase II, the enzyme that transcribes most of the genes in the human genome, operates as part of a large protein complex with multiple regulatory and accessory subunits.[87]

DNA

Genetic recombination

Structure of the Holliday junction intermediate in genetic recombination. The four separate DNA strands are coloured red, blue, green and yellow.[88] A DNA helix usually does not interact with other segments of DNA, and in human cells the different chromosomes even occupy separate areas in the nucleus called "chromosome territories".[89] This physical separation of different chromosomes is important for the ability of DNA to function as a stable repository for information, as one of the few times chromosomes interact is during chromosomal crossover when they recombine. Chromosomal crossover is when two DNA helices break, swap a section and then rejoin. Recombination involves the breakage and rejoining of two chromosomes (M and F) to produce two re-arranged chromosomes (C1 and C2).

Recombination allows chromosomes to exchange genetic information and produces new combinations of genes, which increases the efficiency of natural selection and can be important in the rapid evolution of [90] new proteins. Genetic recombination can also be involved in DNA repair, particularly in the cell's response to double-strand breaks.[91] The most common form of chromosomal crossover is homologous recombination, where the two chromosomes involved share very similar sequences. Non-homologous recombination can be damaging to cells, as it can produce chromosomal translocations and genetic abnormalities. The recombination reaction is catalyzed by enzymes known as recombinases, such as RAD51.[92] The first step in recombination is a double-stranded break caused by either an endonuclease or damage to the DNA.[93] A series of steps catalyzed in part by the recombinase then leads to joining of the two helices by at least one Holliday junction, in which a segment of a single strand in each helix is annealed to the complementary strand in the other helix. The Holliday junction is a tetrahedral junction structure that can be moved along the pair of chromosomes, swapping one strand for another. The recombination reaction is then halted by cleavage of the junction and re-ligation of the released DNA.[94]

DNA

Evolution DNA contains the genetic information that allows all modern living things to function, grow and reproduce. However, it is unclear how long in the 4-billion-year history of life DNA has performed this function, as it has been proposed that the earliest forms of life may have used RNA as their genetic material.[][95] RNA may have acted as the central part of early cell metabolism as it can both transmit genetic information and carry out catalysis as part of ribozymes.[96] This ancient RNA world where nucleic acid would have been used for both catalysis and genetics may have influenced the evolution of the current genetic code based on four nucleotide bases. This would occur, since the number of different bases in such an organism is a trade-off between a small number of bases increasing replication accuracy and a large number of bases increasing the catalytic efficiency of ribozymes.[97] However, there is no direct evidence of ancient genetic systems, as recovery of DNA from most fossils is impossible. This is because DNA survives in the environment for less than one million years, and slowly degrades into short fragments in solution.[98] Claims for older DNA have been made, most notably a report of the isolation of a viable bacterium from a salt crystal 250 million years old,[99] but these claims are controversial.[100][101] On 8 August 2011, a report, based on NASA studies with meteorites found on Earth, was published suggesting building blocks of DNA (adenine, guanine and related organic molecules) may have been formed extraterrestrially in outer space.[][][]

Uses in technology Genetic engineering Methods have been developed to purify DNA from organisms, such as phenol-chloroform extraction, and to manipulate it in the laboratory, such as restriction digests and the polymerase chain reaction. Modern biology and biochemistry make intensive use of these techniques in recombinant DNA technology. Recombinant DNA is a man-made DNA sequence that has been assembled from other DNA sequences. They can be transformed into organisms in the form of plasmids or in the appropriate format, by using a viral vector.[102] The genetically modified organisms produced can be used to produce products such as recombinant proteins, used in medical research,[103] or be grown in agriculture.[104][105]

Forensics Forensic scientists can use DNA in blood, semen, skin, saliva or hair found at a crime scene to identify a matching DNA of an individual, such as a perpetrator. This process is formally termed DNA profiling, but may also be called "genetic fingerprinting". In DNA profiling, the lengths of variable sections of repetitive DNA, such as short tandem repeats and minisatellites, are compared between people. This method is usually an extremely reliable technique for identifying a matching DNA.[106] However, identification can be complicated if the scene is contaminated with DNA from several people.[107] DNA profiling was developed in 1984 by British geneticist Sir Alec Jeffreys,[108] and first used in forensic science to convict Colin Pitchfork in the 1988 Enderby murders case.[109] The development of forensic science, and the ability to now obtain genetic matching on minute samples of blood, skin, saliva or hair has led to a re-examination of a number of cases. Evidence can now be uncovered that was not scientifically possible at the time of the original examination. Combined with the removal of the double jeopardy law in some places, this can allow cases to be reopened where previous trials have failed to produce sufficient evidence to convince a jury. People charged with serious crimes may be required to provide a sample of DNA for matching purposes. The most obvious defence to DNA matches obtained forensically is to claim that cross-contamination of evidence has taken place. This has resulted in meticulous strict handling procedures with new cases of serious crime. DNA profiling is also used to identify victims of mass casualty incidents.[110] As well as positively identifying bodies or body parts in serious accidents, DNA profiling is being successfully used to identify individual victims in mass war graves– matching to family members.

DNA

Bioinformatics Bioinformatics involves the manipulation, searching, and data mining of biological data, and this includes DNA sequence data. The development of techniques to store and search DNA sequences have led to widely applied advances in computer science, especially string searching algorithms, machine learning and database theory.[111] String searching or matching algorithms, which find an occurrence of a sequence of letters inside a larger sequence of letters, were developed to search for specific sequences of nucleotides.[112] The DNA sequence may be aligned with other DNA sequences to identify homologous sequences and locate the specific mutations that make them distinct. These techniques, especially multiple sequence alignment, are used in studying phylogenetic relationships and protein function.[113] Data sets representing entire genomes' worth of DNA sequences, such as those produced by the Human Genome Project, are difficult to use without the annotations that identify the locations of genes and regulatory elements on each chromosome. Regions of DNA sequence that have the characteristic patterns associated with protein- or RNA-coding genes can be identified by gene finding algorithms, which allow researchers to predict the presence of particular gene products and their possible functions in an organism even before they have been isolated experimentally.[] Entire genomes may also be compared, which can shed light on the evolutionary history of particular organism and permit the examination of complex evolutionary events.

DNA nanotechnology DNA nanotechnology uses the unique molecular recognition properties of DNA and other nucleic acids to create self-assembling branched DNA complexes with useful properties.[115] DNA is thus used as a structural material rather than as a carrier of biological information. This has led to the creation of two-dimensional periodic lattices (both tile-based as well as using the "DNA origami" method) as well as three-dimensional The DNA structure at left (schematic shown) will self-assemble into the structure structures in the shapes of visualized by atomic force microscopy at right. DNA nanotechnology is the field that seeks to design nanoscale structures using the molecular recognition properties of DNA polyhedra.[116] Nanomechanical [114] molecules. Image from Strong, 2004 . devices and algorithmic self-assembly have also been demonstrated,[117] and these DNA structures have been used to template the arrangement of other molecules such as gold nanoparticles and streptavidin proteins.[118]

History and anthropology Because DNA collects mutations over time, which are then inherited, it contains historical information, and, by comparing DNA sequences, geneticists can infer the evolutionary history of organisms, their phylogeny.[119] This field of phylogenetics is a powerful tool in evolutionary biology. If DNA sequences within a species are compared, population geneticists can learn the history of particular populations. This can be used in studies ranging from ecological genetics to anthropology; For example, DNA evidence is being used to try to identify the Ten Lost Tribes of Israel.[120][121] DNA has also been used to look at modern family relationships, such as establishing family relationships between the descendants of Sally Hemings and Thomas Jefferson. This usage is closely related to the use of DNA in criminal

DNA

investigations detailed above. Indeed, some criminal investigations have been solved when DNA from crime scenes has matched relatives of the guilty individual.[122]

Information storage In a paper published in Nature in January, 2013, scientists from the European Bioinformatics Institute and Agilent Technologies proposed a mechanism to use DNA's ability to code information as a means of digital data storage. The group was able to encode 739 kilobytes of data into DNA code, synthesize the actual DNA, then sequence the DNA and decode the information back to its original form, with a reported 100% accuracy. The encoded information consisted of text files and audio files. A prior experiment was published in August 2012. It was conducted by researchers at Harvard University, where the text of a 54,000-word book was encoded in DNA.[123][124]

History of DNA research DNA was first isolated by the Swiss physician Friedrich Miescher who, in 1869, discovered a microscopic substance in the pus of discarded surgical bandages. As it resided in the nuclei of cells, he called it "nuclein".[125] In 1878, Albrecht Kossel isolated the non-protein component of "nuclein", nucleic acid, and later isolated its five primary nucleobases.[] In 1919, Phoebus Levene identified the base, sugar and phosphate nucleotide unit.[126] Levene suggested that DNA consisted of a string of nucleotide units linked together through the phosphate groups. However, Levene thought the chain was short and the bases repeated in a fixed order. In 1937 William Astbury produced the first X-ray diffraction patterns that showed that DNA had a regular structure.[127]

James Watson and Francis Crick (right), co-originators of the double-helix model, with Maclyn McCarty (left).

In 1927, Nikolai Koltsov proposed that inherited traits would be inherited via a "giant hereditary molecule" made up of "two mirror strands that would replicate in a semi-conservative fashion using each strand as a template".[] In 1928, Frederick Griffith discovered that traits of the "smooth" form of Pneumococcus could be transferred to the "rough" form of the same bacteria by mixing killed "smooth" bacteria with the live "rough" form.[128] This system provided the first clear suggestion that DNA carries genetic information—the Avery–MacLeod–McCarty experiment—when Oswald Avery, along with coworkers Colin MacLeod and Maclyn McCarty, identified DNA as the transforming principle in 1943.[129] DNA's role in heredity was confirmed in 1952, when Alfred Hershey and Martha Chase in the Hershey–Chase experiment showed that DNA is the genetic material of the T2 phage.[130] In 1953, James Watson and Francis Crick suggested what is now accepted as the first correct double-helix model of DNA structure in the journal Nature.[] Their double-helix, molecular model of DNA was then based on a single X-ray diffraction image (labeled as "Photo 51")[131] taken by Rosalind Franklin and Raymond Gosling in May 1952, as well as the information that the DNA bases are paired— also obtained through private communications from Erwin Chargaff in the previous years. Chargaff's rules played a very important role in establishing double-helix configurations for B-DNA as well as A-DNA. Experimental evidence supporting the Watson and Crick model was published in a series of five articles in the same issue of Nature.[132] Of these, Franklin and Gosling's paper was the first publication of their own X-ray diffraction data and original analysis method that partially supported the Watson and Crick model;[][133] this issue also contained an article on DNA structure by Maurice Wilkins and two of his colleagues, whose analysis and in vivo B-DNA X-ray patterns also supported the presence in vivo of the double-helical DNA configurations as proposed by Crick and Watson for their double-helix molecular model of DNA in the previous two pages of Nature.[] In 1962, after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine.[134]

DNA Nobel Prizes were awarded only to living recipients at the time. A debate continues about who should receive credit for the discovery.[135] In an influential presentation in 1957, Crick laid out the central dogma of molecular biology, which foretold the relationship between DNA, RNA, and proteins, and articulated the "adaptor hypothesis".[136] Final confirmation of the replication mechanism that was implied by the double-helical structure followed in 1958 through the Meselson–Stahl experiment.[137] Further work by Crick and coworkers showed that the genetic code was based on non-overlapping triplets of bases, called codons, allowing Har Gobind Khorana, Robert W. Holley and Marshall Warren Nirenberg to decipher the genetic code.[138] These findings represent the birth of molecular biology.

References [3] pp. 14–15. [6] Berg J., Tymoczko J. and Stryer L. (2002) Biochemistry. W. H. Freeman and Company ISBN 0-7167-4955-6 [7] Abbreviations and Symbols for Nucleic Acids, Polynucleotides and their Constituents (http:/ / www. chem. qmul. ac. uk/ iupac/ misc/ naabb. html) IUPAC-IUB Commission on Biochemical Nomenclature (CBN). Retrieved 3 January 2006. [8] Created from PDB 1D65 (http:/ / www. rcsb. org/ pdb/ cgi/ explore. cgi?pdbId=1D65) [15] Designation of the two strands of DNA (http:/ / www. chem. qmul. ac. uk/ iubmb/ newsletter/ misc/ DNA. html) JCBN/NC-IUB Newsletter 1989. Retrieved 7 May 2008 [25] http:/ / cogprints. org/ 3822/ [26] Hosemann R., Bagchi R.N., Direct analysis of diffraction by matter, North-Holland Publs., Amsterdam– New York, 1962. [34] Created from NDB UD0017 (http:/ / ndbserver. rutgers. edu/ atlas/ xray/ structures/ U/ ud0017/ ud0017. html) [38] Hu Q, Rosenfeld MG. (2012) Epigenetic regulation of human embryonic stem cells. Front Genet. 3:238. doi: 10.3389/fgene.2012.00238. PMID 23133442 [45] Created from PDB 1JDG (http:/ / www. rcsb. org/ pdb/ cgi/ explore. cgi?pdbId=1JDG) [51] Bernstein H, Payne CM, Bernstein C, Garewal H, Dvorak K. (2008) Cancer and aging as consequences of un-repaired DNA damage. In: New Research on DNA Damage (Editors: Honoka Kimura And Aoi Suzuki) Nova Science Publishers, Inc., New York, Chapter 1, pp. 1–47. ISBN 978-1-60456-581-2 [63] Created from PDB 1MSW (http:/ / www. rcsb. org/ pdb/ explore/ explore. do?structureId=1MSW) [76] Created from PDB 1LMB (http:/ / www. rcsb. org/ pdb/ explore/ explore. do?structureId=1LMB) [80] Created from PDB 1RVA (http:/ / www. rcsb. org/ pdb/ explore/ explore. do?structureId=1RVA) [88] Created from PDB 1M6G (http:/ / www. rcsb. org/ pdb/ explore/ explore. do?structureId=1M6G) [109] Colin Pitchfork— first murder conviction on DNA evidence also clears the prime suspect (http:/ / web. archive. org/ web/ 20061214004903/ http:/ / www. forensic. gov. uk/ forensic_t/ inside/ news/ list_casefiles. php?case=1) Forensic Science Service Accessed 23 December 2006 [112] Gusfield, Dan. Algorithms on Strings, Trees, and Sequences: Computer Science and Computational Biology. Cambridge University Press, 15 January 1997. ISBN 978-0-521-58519-4. [114] http:/ / dx. doi. org/ 10. 1371/ journal. pbio. 0020073 [120] Lost Tribes of Israel, NOVA, PBS airdate: 22 February 2000. Transcript available from PBS.org (http:/ / www. pbs. org/ wgbh/ nova/ transcripts/ 2706israel. html). Retrieved 4 March 2006. [121] Kleiman, Yaakov. "The Cohanim/DNA Connection: The fascinating story of how DNA studies confirm an ancient biblical tradition". (http:/ / www. aish. com/ societywork/ sciencenature/ the_cohanim_-_dna_connection. asp) aish.com (13 January 2000). Retrieved 4 March 2006. [122] Bhattacharya, Shaoni. "Killer convicted thanks to relative's DNA". (http:/ / www. newscientist. com/ article. ns?id=dn4908) newscientist.com (20 April 2004). Retrieved 22 December 06. [131] The B-DNA X-ray pattern on the right of this linked image (http:/ / osulibrary. oregonstate. edu/ specialcollections/ coll/ pauling/ dna/ pictures/ sci9. 001. 5. html) was obtained by Rosalind Franklin and Raymond Gosling in May 1952 at high hydration levels of DNA and it has been labeled as "Photo 51" [132] Nature Archives Double Helix of DNA: 50 Years (http:/ / www. nature. com/ nature/ dna50/ archive. html) [134] The Nobel Prize in Physiology or Medicine 1962 (http:/ / nobelprize. org/ nobel_prizes/ medicine/ laureates/ 1962/ ) Nobelprize .org Accessed 22 December 06 [136] Crick, F.H.C. On degenerate templates and the adaptor hypothesis (PDF). (http:/ / genome. wellcome. ac. uk/ assets/ wtx030893. pdf) genome.wellcome.ac.uk (Lecture, 1955). Retrieved 22 December 2006. [138] The Nobel Prize in Physiology or Medicine 1968 (http:/ / nobelprize. org/ nobel_prizes/ medicine/ laureates/ 1968/ ) Nobelprize.org Accessed 22 December 06

DNA

Further reading • Berry, Andrew; Watson, James. (2003). DNA: the secret of life. New York: Alfred A. Knopf. ISBN0-375-41546-7. • Calladine, Chris R.; Drew, Horace R.; Luisi, Ben F. and Travers, Andrew A. (2003). Understanding DNA: the molecule & how it works. Amsterdam: Elsevier Academic Press. ISBN0-12-155089-3. • Dennis, Carina; Julie Clayton (2003). 50 years of DNA. Basingstoke: Palgrave Macmillan. ISBN1-4039-1479-6. • Judson, Horace F. 1979. The Eighth Day of Creation: Makers of the Revolution in Biology. Touchstone Books, ISBN 0-671-22540-5. 2nd edition: Cold Spring Harbor Laboratory Press, 1996 paperback: ISBN 0-87969-478-5. • Olby, Robert C. (1994). The path to the double helix: the discovery of DNA. New York: Dover Publications. ISBN0-486-68117-3., first published in October 1974 by MacMillan, with foreword by Francis Crick;the definitive DNA textbook,revised in 1994 with a 9 page postscript • Micklas, David. 2003. DNA Science: A First Course. Cold Spring Harbor Press: ISBN 978-0-87969-636-8 • Ridley, Matt (2006). Francis Crick: discoverer of the genetic code. Ashland, OH: Eminent Lives, Atlas Books. ISBN0-06-082333-X. • Olby, Robert C. (2009). Francis Crick: A Biography. Plainview, N.Y: Cold Spring Harbor Laboratory Press. ISBN0-87969-798-9. • Rosenfeld, Israel. 2010. DNA: A Graphic Guide to the Molecule that Shook the World. Columbia University Press: ISBN 978-0-231-14271-7 • Schultz, Mark and Zander Cannon. 2009. The Stuff of Life: A Graphic Guide to Genetics and DNA. Hill and Wang: ISBN 0-8090-8947-5 • Stent, Gunther Siegmund; Watson, James. (1980). The double helix: a personal account of the discovery of the structure of DNA. New York: Norton. ISBN0-393-95075-1. • Watson, James. 2004. DNA: The Secret of Life. Random House: ISBN 978-0-09-945184-6 • Wilkins, Maurice (2003). The third man of the double helix the autobiography of Maurice Wilkins. Cambridge, Eng: University Press. ISBN0-19-860665-6.

External links • Books about DNA: Online books (http://onlinebooks.library.upenn.edu/webbin/ftl?st=&su=DNA&library=OLBP), Resources in your library (http://onlinebooks.library.upenn.edu/webbin/ftl?st=&su=DNA), Resources in other libraries (http:/ / onlinebooks. library. upenn. edu/ webbin/ ftl?st=& su=DNA& library=0CHOOSE0) • DNA (http://www.dmoz.org/Science/Biology/Biochemistry_and_Molecular_Biology/Biomolecules/ Nucleic_Acids/DNA//) at the Open Directory Project • DNA binding site prediction on protein (http://pipe.scs.fsu.edu/displar.html) • DNA the Double Helix Game (http://nobelprize.org/educational_games/medicine/dna_double_helix/) From the official Nobel Prize web site • DNA under electron microscope (http://www.fidelitysystems.com/Unlinked_DNA.html) • Dolan DNA Learning Center (http://www.dnalc.org/) • Double Helix: 50 years of DNA (http://www.nature.com/nature/dna50/archive.html), Nature • Proteopedia DNA (http://www.proteopedia.org/wiki/index.php/DNA) • ENCODE threads explorer (http://www.nature.com/encode/) ENCODE Home page. Nature (journal) • Double Helix 1953–2003 (http://www.ncbe.reading.ac.uk/DNA50/) National Centre for Biotechnology Education

DNA • Genetic Education Modules for Teachers (http://www.genome.gov/10506718)—DNA from the Beginning Study Guide • PDB Molecule of the Month pdb23_1 (http://www.rcsb.org/pdb/static.do?p=education_discussion/ molecule_of_the_month/pdb23_1.html) • Rosalind Franklin's contributions to the study of DNA (http://mason.gmu.edu/~emoody/rfranklin.html) • U.S. National DNA Day (http://www.genome.gov/10506367)—watch videos and participate in real-time chat with top scientists • Clue to chemistry of heredity found (http://www.nytimes.com/packages/pdf/science/dna-article.pdf) The New York Times June 1953. First American newspaper coverage of the discovery of the DNA structure • Olby R (2003). "Quiet debut for the double helix". Nature 421 (6921): 402–5. doi: 10.1038/nature01397 (http:// dx.doi.org/10.1038/nature01397). PMID 12540907 (http://www.ncbi.nlm.nih.gov/pubmed/12540907). • DNA from the Beginning (http://www.dnaftb.org/) Another DNA Learning Center site on DNA, genes, and heredity from Mendel to the human genome project. • The Register of Francis Crick Personal Papers 1938– 2007 (http://orpheus.ucsd.edu/speccoll/testing/html/ mss0660a.html#abstract) at Mandeville Special Collections Library, University of California, San Diego • Seven-page, handwritten letter that Crick sent to his 12-year-old son Michael in 1953 describing the structure of DNA. (http://www.nature.com/polopoly_fs/7.9746!/file/Crick letter to Michael.pdf) See Crick’s medal goes under the hammer (http://www.nature.com/news/crick-s-medal-goes-under-the-hammer-1.12705), Nature, 5 April 2013.

Proteins and amino acids Protein Proteins (pron.: /ˈproʊˌtiːnz/ or /ˈproʊti.ɨnz/) are large biological molecules consisting of one or more chains of amino acids. Proteins perform a vast array of functions within living organisms, including catalyzing metabolic reactions, replicating DNA, responding to stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in folding of the protein into a specific three-dimensional structure that determines its activity. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The sequence of amino acids in a protein A representation of the 3D structure of the protein myoglobin showing turquoise alpha helices. This is defined by the sequence of a gene, which is encoded in the genetic protein was the first to have its structure solved code. In general, the genetic code specifies 20 standard amino acids; by X-ray crystallography. Towards the however, in certain organisms the genetic code can include right-center among the coils, a prosthetic group selenocysteine and—in certain archaea—pyrrolysine. Shortly after or called a heme group (shown in gray) with a bound oxygen molecule (red). even during synthesis, the residues in a protein are often chemically modified by posttranslational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can also work together to achieve a particular function, and they often associate to form stable protein complexes. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in virtually every process within cells. Many proteins are enzymes that catalyze biochemical reactions and are vital to metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape. Other proteins are important in cell signaling, immune responses, cell adhesion, and the cell cycle. Proteins are also necessary in animals' diets, since animals cannot synthesize all the amino acids they need and must obtain essential amino acids from food. Through the process of digestion, animals break down ingested protein into free amino acids that are then used in metabolism. Proteins may be purified from other cellular components using a variety of techniques such as ultracentrifugation, precipitation, electrophoresis, and chromatography; the advent of genetic engineering has made possible a number of methods to facilitate purification. Methods commonly used to study protein structure and function include immunohistochemistry, site-directed mutagenesis, nuclear magnetic resonance and mass spectrometry.

Protein

Biochemistry Most proteins consist of linear polymers built from series of up to 20 different L-α-amino acids. All proteinogenic amino acids possess common structural features, including an α-carbon to which an amino group, a carboxyl group, and a variable side chain are bonded. Only proline differs from this basic structure as it contains an unusual ring to the N-end amine group, which forces the CO–NH amide moiety into a fixed conformation.[] The side chains of the standard amino acids, detailed in the list of standard amino acids, have a great variety of chemical structures and properties; it is the combined effect of all of the amino acid side chains in a protein that ultimately determines its three-dimensional structure and its chemical reactivity.[] The amino acids in a polypeptide chain are linked by peptide bonds. Once linked in the protein chain, an individual amino acid is called a residue, and the linked series of carbon, nitrogen, and oxygen atoms are known as the main chain or protein backbone.[1]

Chemical structure of the peptide bond (bottom) and the three-dimensional structure of a peptide bond between an alanine and an adjacent amino acid (top/inset)

Resonance structures of the peptide bond that links individual amino acids to form a protein polymer

The peptide bond has two resonance forms that contribute some double-bond character and inhibit rotation around its axis, so that the alpha carbons are roughly coplanar. The other two dihedral angles in the peptide bond determine the local shape assumed by the protein backbone.[2] The end of the protein with a free carboxyl group is known as the C-terminus or carboxy terminus, whereas the end with a free amino group is known as the N-terminus or amino terminus. The words protein, polypeptide, and peptide are a little ambiguous and can overlap in meaning. Protein is generally used to refer to the complete biological molecule in a stable conformation, whereas peptide is generally reserved for a short amino acid oligomers often lacking a stable three-dimensional structure. However, the boundary between the two is not well defined and usually lies near 20–30 residues.[] Polypeptide can refer to any single linear chain of amino acids, usually regardless of length, but often implies an absence of a defined conformation.

Protein

Synthesis Proteins are assembled from amino acids using information encoded in genes. Each protein has its own unique amino acid sequence that is specified by the nucleotide sequence of the gene encoding this protein. The genetic code is a set of three-nucleotide sets called codons and each three-nucleotide combination designates an amino acid, for example AUG (adenine-uracil-guanine) is the code for methionine. Because DNA contains four nucleotides, the total number of possible codons is 64; hence, there is some redundancy in the genetic code, with some amino acids specified by more than one codon.[3] Genes encoded A ribosome produces a protein using mRNA as template. in DNA are first transcribed into pre-messenger RNA (mRNA) by proteins such as RNA polymerase. Most organisms then process the pre-mRNA (also known as a primary transcript) using various forms of Post-transcriptional modification to form the mature mRNA, which is then used as a template for protein synthesis by the ribosome. In prokaryotes the mRNA may either be used as soon as it is produced, or be bound by a ribosome after having moved away from the nucleoid. In contrast, eukaryotes make mRNA in the cell nucleus and then The DNA sequence of a gene encodes the amino acid sequence of a protein. translocate it across the nuclear membrane into the cytoplasm, where protein synthesis then takes place. The rate of protein synthesis is higher in prokaryotes than eukaryotes and can reach up to 20 amino acids per second.[] The process of synthesizing a protein from an mRNA template is known as translation. The mRNA is loaded onto the ribosome and is read three nucleotides at a time by matching each codon to its base pairing anticodon located on a transfer RNA molecule, which carries the amino acid corresponding to the codon it recognizes. The enzyme aminoacyl tRNA synthetase "charges" the tRNA molecules with the correct amino acids. The growing polypeptide is often termed the nascent chain. Proteins are always biosynthesized from N-terminus to C-terminus.[3] The size of a synthesized protein can be measured by the number of amino acids it contains and by its total molecular mass, which is normally reported in units of daltons (synonymous with atomic mass units), or the derivative unit kilodalton (kDa). Yeast proteins are on average 466 amino acids long and 53 kDa in mass.[] The largest known proteins are the titins, a component of the muscle sarcomere, with a molecular mass of almost 3,000 kDa and a total length of almost 27,000 amino acids.[]

Chemical synthesis Short proteins can also be synthesized chemically by a family of methods known as peptide synthesis, which rely on organic synthesis techniques such as chemical ligation to produce peptides in high yield.[] Chemical synthesis allows for the introduction of non-natural amino acids into polypeptide chains, such as attachment of fluorescent probes to amino acid side chains.[] These methods are useful in laboratory biochemistry and cell biology, though generally not for commercial applications. Chemical synthesis is inefficient for polypeptides longer than about 300 amino acids, and the synthesized proteins may not readily assume their native tertiary structure. Most chemical synthesis methods proceed from C-terminus to N-terminus, opposite the biological reaction.[]

Protein

Structure Mostproteins fold into unique 3-dimensional structures. The shape into which a protein naturally folds is known as its native conformation.[4] Although many proteins can fold unassisted, simply through the chemical properties of their amino acids, others require the aid of molecular chaperones to fold into their native states.[5] Biochemists often refer to four distinct aspects of a protein's structure:[6]

The crystal structure of the chaperonin. Chaperonins assist protein folding.

• Primary structure: the amino acid sequence. • Secondary structure: regularly repeating local structures stabilized by hydrogen bonds. The most common examples are the alpha helix, beta sheet and turns. Because secondary structures are local, many regions of different secondary structure can be present in the same protein molecule.

Three possible representations of the three-dimensional structure of the protein triose phosphate isomerase. Left: all-atom representation colored by atom type. Middle: Simplified representation illustrating the backbone conformation, colored by secondary structure. Right: Solvent-accessible surface representation colored by residue type (acidic residues red, basic residues blue, polar residues green, nonpolar residues white)

• Tertiary structure: the overall shape of a single protein molecule; the spatial relationship of the secondary structures to one another. Tertiary structure is generally stabilized by nonlocal interactions, most commonly the formation of a hydrophobic core, but also through salt bridges, hydrogen bonds, disulfide bonds, and even posttranslational modifications. The term "tertiary structure" is often used as synonymous with the term fold. The tertiary structure is what controls the basic function of the protein. • Quaternary structure: the structure formed by several protein molecules (polypeptide chains), usually called protein subunits in this context, which function as a single protein complex.

Proteins are not entirely rigid molecules. In addition to these levels of structure, proteins may shift between several related structures while they perform their functions. In the context of these functional rearrangements, these tertiary or quaternary structures are usually referred to as "conformations", and transitions between them are called conformational changes. Such changes are often induced by the binding of a substrate molecule to an enzyme's active site, or the physical region of the protein that participates in chemical catalysis. In solution proteins also undergo variation in structure through thermal vibration and the collision with other molecules.[7]

Protein

Proteins can be informally divided into three main classes, which correlate with typical tertiary structures: globular proteins, fibrous proteins, and membrane proteins. Almost all globular proteins are soluble and many are enzymes. Fibrous proteins are often Molecular surface of several proteins showing their comparative sizes. From left to structural, such as collagen, the major right are: immunoglobulin G (IgG, an antibody), hemoglobin, insulin (a hormone), adenylate kinase (an enzyme), and glutamine synthetase (an enzyme). component of connective tissue, or keratin, the protein component of hair and nails. Membrane proteins often serve as receptors or provide channels for polar or charged molecules to pass through the cell membrane.[8] A special case of intramolecular hydrogen bonds within proteins, poorly shielded from water attack and hence promoting their own dehydration, are called dehydrons.[]

Structure determination Discovering the tertiary structure of a protein, or the quaternary structure of its complexes, can provide important clues about how the protein performs its function. Common experimental methods of structure determination include X-ray crystallography and NMR spectroscopy, both of which can produce information at atomic resolution. However, NMR experiments are able to provide information from which a subset of distances between pairs of atoms can be estimated, and the final possible conformations for a protein are determined by solving a distance geometry problem. Dual polarisation interferometry is a quantitative analytical method for measuring the overall protein conformation and conformational changes due to interactions or other stimulus. Circular dichroism is another laboratory technique for determining internal beta sheet/ helical composition of proteins. Cryoelectron microscopy is used to produce lower-resolution structural information about very large protein complexes, including assembled viruses;[9] a variant known as electron crystallography can also produce high-resolution information in some cases, especially for two-dimensional crystals of membrane proteins.[] Solved structures are usually deposited in the Protein Data Bank (PDB), a freely available resource from which structural data about thousands of proteins can be obtained in the form of Cartesian coordinates for each atom in the protein.[] Many more gene sequences are known than protein structures. Further, the set of solved structures is biased toward proteins that can be easily subjected to the conditions required in X-ray crystallography, one of the major structure determination methods. In particular, globular proteins are comparatively easy to crystallize in preparation for X-ray crystallography. Membrane proteins, by contrast, are difficult to crystallize and are underrepresented in the PDB.[] Structural genomics initiatives have attempted to remedy these deficiencies by systematically solving representative structures of major fold classes. Protein structure prediction methods attempt to provide a means of generating a plausible structure for proteins whose structures have not been experimentally determined.[]

Cellular functions Proteins are the chief actors within the cell, said to be carrying out the duties specified by the information encoded in genes.[] With the exception of certain types of RNA, most other biological molecules are relatively inert elements upon which proteins act. Proteins make up half the dry weight of an Escherichia coli cell, whereas other macromolecules such as DNA and RNA make up only 3% and 20%, respectively.[10] The set of proteins expressed in a particular cell or cell type is known as its proteome.

Protein

The chief characteristic of proteins that also allows their diverse set of functions is their ability to bind other molecules specifically and tightly. The region of the protein responsible for binding another molecule is known as the binding site and is often a depression or "pocket" on the molecular surface. This binding ability is mediated by the tertiary structure of the protein, which defines the binding site pocket, and by the chemical properties of the surrounding amino acids' side chains. Protein binding can be extraordinarily tight and specific; The enzyme hexokinase is shown as a for example, the ribonuclease inhibitor protein binds to human conventional ball-and-stick molecular model. To −15 angiogenin with a sub-femtomolar dissociation constant (1 M). substrates, ATP and glucose. Extremely minor chemical changes such as the addition of a single methyl group to a binding partner can sometimes suffice to nearly eliminate binding; for example, the aminoacyl tRNA synthetase specific to the amino acid valine discriminates against the very similar side chain of the amino acid isoleucine.[] Proteins can bind to other proteins as well as to small-molecule substrates. When proteins bind specifically to other copies of the same molecule, they can oligomerize to form fibrils; this process occurs often in structural proteins that consist of globular monomers that self-associate to form rigid fibers. Protein–protein interactions also regulate enzymatic activity, control progression through the cell cycle, and allow the assembly of large protein complexes that carry out many closely related reactions with a common biological function. Proteins can also bind to, or even be integrated into, cell membranes. The ability of binding partners to induce conformational changes in proteins allows the construction of enormously complex signaling networks.[11] Importantly, as interactions between proteins are reversible, and depend heavily on the availability of different groups of partner proteins to form aggregates that are capable to carry out discrete sets of function, study of the interactions between specific proteins is a key to understand important aspects of cellular function, and ultimately the properties that distinguish particular cell types.[][]

Enzymes The best-known role of proteins in the cell is as enzymes, which catalyze chemical reactions. Enzymes are usually highly specific and accelerate only one or a few chemical reactions. Enzymes carry out most of the reactions involved in metabolism, as well as manipulating DNA in processes such as DNA replication, DNA repair, and transcription. Some enzymes act on other proteins to add or remove chemical groups in a process known as posttranslational modification. About 4,000 reactions are known to be catalyzed by enzymes.[] The rate acceleration conferred by enzymatic catalysis is often enormous—as much as 1017-fold increase in rate over the uncatalyzed reaction in the case of orotate decarboxylase (78 million years without the enzyme, 18 milliseconds with the enzyme).[] The molecules bound and acted upon by enzymes are called substrates. Although enzymes can consist of hundreds of amino acids, it is usually only a small fraction of the residues that come in contact with the substrate, and an even smaller fraction—three to four residues on average—that are directly involved in catalysis.[] The region of the enzyme that binds the substrate and contains the catalytic residues is known as the active site. Dirigent proteins are members of a class of proteins which dictate the stereochemistry of a compound synthesized by other enzymes.

Protein

Cell signaling and ligand binding Many proteins are involved in the process of cell signaling and signal transduction. Some proteins, such as insulin, are extracellular proteins that transmit a signal from the cell in which they were synthesized to other cells in distant tissues. Others are membrane proteins that act as receptors whose main function is to bind a signaling molecule and induce a biochemical response in the cell. Many receptors have a binding site exposed on the cell surface and an effector domain within the cell, which may have enzymatic activity or may undergo a conformational change detected by other proteins within the cell.[12] Antibodies are protein components of an adaptive immune system whose main function is to bind antigens, or foreign substances in the body, and target them for destruction. Antibodies can be secreted into the extracellular environment or anchored in the membranes of specialized B cells known as plasma cells. Whereas enzymes are limited in their binding affinity for their substrates by the necessity of conducting their reaction, antibodies have no such constraints. An antibody's binding affinity to its target is extraordinarily high.[13]

Ribbon diagram of a mouse antibody against cholera that binds a carbohydrate antigen

Many ligand transport proteins bind particular small biomolecules and transport them to other locations in the body of a multicellular organism. These proteins must have a high binding affinity when their ligand is present in high concentrations, but must also release the ligand when it is present at low concentrations in the target tissues. The canonical example of a ligand-binding protein is haemoglobin, which transports oxygen from the lungs to other organs and tissues in all vertebrates and has close homologs in every biological kingdom.[14] Lectins are sugar-binding proteins which are highly specific for their sugar moieties. Lectins typically play a role in biological recognition phenomena involving cells and proteins.[] Receptors and hormones are highly specific binding proteins. Transmembrane proteins can also serve as ligand transport proteins that alter the permeability of the cell membrane to small molecules and ions. The membrane alone has a hydrophobic core through which polar or charged molecules cannot diffuse. Membrane proteins contain internal channels that allow such molecules to enter and exit the cell. Many ion channel proteins are specialized to select for only a particular ion; for example, potassium and sodium channels often discriminate for only one of the two ions.[15]

Structural proteins Structural proteins confer stiffness and rigidity to otherwise-fluid biological components. Most structural proteins are fibrous proteins; for example, collagen and elastin are critical components of connective tissue such as cartilage, and keratin is found in hard or filamentous structures such as hair, nails, feathers, hooves, and some animal shells.[16] Some globular proteins can also play structural functions, for example, actin and tubulin are globular and soluble as monomers, but polymerize to form long, stiff fibers that make up the cytoskeleton, which allows the cell to maintain its shape and size. Other proteins that serve structural functions are motor proteins such as myosin, kinesin, and dynein, which are capable of generating mechanical forces. These proteins are crucial for cellular motility of single celled organisms and the sperm of many multicellular organisms which reproduce sexually. They also generate the forces exerted by contracting muscles[17] and play essential roles in intracellular transport.

Protein

Methods of study As some of the most commonly studied biological molecules, the activities and structures of proteins are examined both in vitro and in vivo. In vitro studies of purified proteins in controlled environments are useful for learning how a protein carries out its function: for example, enzyme kinetics studies explore the chemical mechanism of an enzyme's catalytic activity and its relative affinity for various possible substrate molecules. By contrast, in vivo experiments on proteins' activities within cells or even within whole organisms can provide complementary information about where a protein functions and how it is regulated.

Protein purification To perform in vitro analysis, a protein must be purified away from other cellular components. This process usually begins with cell lysis, in which a cell's membrane is disrupted and its internal contents released into a solution known as a crude lysate. The resulting mixture can be purified using ultracentrifugation, which fractionates the various cellular components into fractions containing soluble proteins; membrane lipids and proteins; cellular organelles, and nucleic acids. Precipitation by a method known as salting out can concentrate the proteins from this lysate. Various types of chromatography are then used to isolate the protein or proteins of interest based on properties such as molecular weight, net charge and binding affinity.[18] The level of purification can be monitored using various types of gel electrophoresis if the desired protein's molecular weight and isoelectric point are known, by spectroscopy if the protein has distinguishable spectroscopic features, or by enzyme assays if the protein has enzymatic activity. Additionally, proteins can be isolated according their charge using electrofocusing.[] For natural proteins, a series of purification steps may be necessary to obtain protein sufficiently pure for laboratory applications. To simplify this process, genetic engineering is often used to add chemical features to proteins that make them easier to purify without affecting their structure or activity. Here, a "tag" consisting of a specific amino acid sequence, often a series of histidine residues (a "His-tag"), is attached to one terminus of the protein. As a result, when the lysate is passed over a chromatography column containing nickel, the histidine residues ligate the nickel and attach to the column while the untagged components of the lysate pass unimpeded. A number of different tags have been developed to help researchers purify specific proteins from complex mixtures.[]

Protein

Cellular localization The study of proteins in vivo is often concerned with the synthesis and localization of the protein within the cell. Although many intracellular proteins are synthesized in the cytoplasm and membrane-bound or secreted proteins in the endoplasmic reticulum, the specifics of how proteins are targeted to specific organelles or cellular structures is often unclear. A useful technique for assessing cellular localization uses genetic engineering to express in a cell a fusion protein or chimera consisting of the natural protein of interest linked to a "reporter" such as green fluorescent protein (GFP).[] The fused protein's position within the cell can be cleanly and efficiently visualized using microscopy,[] as shown in the figure opposite. Other methods for elucidating the cellular location of proteins requires the use of known compartmental markers for regions such as the ER, the Golgi, lysosomes or vacuoles, mitochondria, chloroplasts, plasma membrane, etc. With the use of fluorescently tagged versions Proteins in different cellular compartments and structures tagged with green of these markers or of antibodies to known fluorescent protein (here, white) markers, it becomes much simpler to identify the localization of a protein of interest. For example, indirect immunofluorescence will allow for fluorescence colocalization and demonstration of location. Fluorescent dyes are used to label cellular compartments for a similar purpose.[] Other possibilities exist, as well. For example, immunohistochemistry usually utilizes an antibody to one or more proteins of interest that are conjugated to enzymes yielding either luminescent or chromogenic signals that can be compared between samples, allowing for localization information. Another applicable technique is cofractionation in sucrose (or other material) gradients using isopycnic centrifugation.[] While this technique does not prove colocalization of a compartment of known density and the protein of interest, it does increase the likelihood, and is more amenable to large-scale studies. Finally, the gold-standard method of cellular localization is immunoelectron microscopy. This technique also uses an antibody to the protein of interest, along with classical electron microscopy techniques. The sample is prepared for normal electron microscopic examination, and then treated with an antibody to the protein of interest that is conjugated to an extremely electro-dense material, usually gold. This allows for the localization of both ultrastructural details as well as the protein of interest.[] Through another genetic engineering application known as site-directed mutagenesis, researchers can alter the protein sequence and hence its structure, cellular localization, and susceptibility to regulation. This technique even allows the incorporation of unnatural amino acids into proteins, using modified tRNAs,[] and may allow the rational design of new proteins with novel properties.[]

Protein

Proteomics and bioinformatics The total complement of proteins present at a time in a cell or cell type is known as its proteome, and the study of such large-scale data sets defines the field of proteomics, named by analogy to the related field of genomics. Key experimental techniques in proteomics include 2D electrophoresis,[] which allows the separation of a large number of proteins, mass spectrometry,[] which allows rapid high-throughput identification of proteins and sequencing of peptides (most often after in-gel digestion), protein microarrays,[] which allow the detection of the relative levels of a large number of proteins present in a cell, and two-hybrid screening, which allows the systematic exploration of protein–protein interactions.[] The total complement of biologically possible such interactions is known as the interactome.[] A systematic attempt to determine the structures of proteins representing every possible fold is known as structural genomics.[] The large amount of genomic and proteomic data available for a variety of organisms, including the human genome, allows researchers to efficiently identify homologous proteins in distantly related organisms by sequence alignment. Sequence profiling tools can perform more specific sequence manipulations such as restriction enzyme maps, open reading frame analyses for nucleotide sequences, and secondary structure prediction. From this data phylogenetic trees can be constructed and evolutionary hypotheses developed using special software like ClustalW regarding the ancestry of modern organisms and the genes they express. The field of bioinformatics seeks to assemble, annotate, and analyze genomic and proteomic data, applying computational techniques to biological problems such as gene finding and cladistics.

Structure prediction and simulation Complementary to the field of structural genomics, protein structure prediction seeks to develop efficient ways to provide plausible models for proteins whose structures have not yet been determined experimentally.[] The most successful type of structure prediction, known as homology modeling, relies on the existence of a "template" structure with sequence similarity to the protein being modeled; structural genomics' goal is to provide sufficient representation in solved structures to model most of those that remain.[] Although producing accurate models remains a challenge when only distantly related template structures are available, it has been suggested that sequence alignment is the bottleneck in this process, as quite accurate models can be produced if a "perfect" sequence alignment is known.[] Many structure prediction methods have served to inform the emerging field of protein engineering, in which novel protein folds have already been designed.[] A more complex computational problem is the prediction of intermolecular interactions, such as in molecular docking and protein–protein interaction prediction.[] The processes of protein folding and binding can be simulated using such technique as molecular mechanics, in particular, molecular dynamics and Monte Carlo, which increasingly take advantage of parallel and distributed computing (Folding@home project;[] molecular modeling on GPU). The folding of small alpha-helical protein domains such as the villin headpiece[] and the HIV accessory protein[] have been successfully simulated in silico, and hybrid methods that combine standard molecular dynamics with quantum mechanics calculations have allowed exploration of the electronic states of rhodopsins.[]

Nutrition Most microorganisms and plants can biosynthesize all 20 standard amino acids, while animals (including humans) must obtain some of the amino acids from the diet.[10] The amino acids that an organism cannot synthesize on its own are referred to as essential amino acids. Key enzymes that synthesize certain amino acids are not present in animals— such as aspartokinase, which catalyzes the first step in the synthesis of lysine, methionine, and threonine from aspartate. If amino acids are present in the environment, microorganisms can conserve energy by taking up the amino acids from their surroundings and downregulating their biosynthetic pathways.

Protein

In animals, amino acids are obtained through the consumption of foods containing protein. Ingested proteins are then broken down into amino acids through digestion, which typically involves denaturation of the protein through exposure to acid and hydrolysis by enzymes called proteases. Some ingested amino acids are used for protein biosynthesis, while others are converted to glucose through gluconeogenesis, or fed into the citric acid cycle. This use of protein as a fuel is particularly important under starvation conditions as it allows the body's own proteins to be used to support life, particularly those found in muscle.[] Amino acids are also an important dietary source of nitrogen.[citation needed]

History and etymology Proteins were recognized as a distinct class of biological molecules in the eighteenth century by Antoine Fourcroy and others, distinguished by the molecules' ability to coagulate or flocculate under treatments with heat or acid[citation needed] . Noted examples at the time included albumin from egg whites, blood serum albumin, fibrin, and wheat gluten. Proteins were first described by the Dutch chemist Gerardus Johannes Mulder and named by the Swedish chemist Jöns Jacob Berzelius in 1838. Mulder carried out elemental analysis of common proteins and found that nearly all proteins had the same empirical formula, C400H620N100O120P1S1.[] He came to the erroneous conclusion that they might be composed of a single type of (very large) molecule. The term "protein" to describe these molecules was proposed by Mulder's associate Berzelius; protein is derived from the Greek word πρωτεῖος (proteios), meaning "primary",[19] "in the lead", or "standing in front".[] Mulder went on to identify the products of protein degradation such as the amino acid leucine for which he found a (nearly correct) molecular weight of 131 Da.[] Early nutritional scientists such as the German Carl von Voit believed that protein was the most important nutrient for maintaining the structure of the body, because it was generally believed that "flesh makes flesh."[] The central role of proteins as enzymes in living organisms was not fully appreciated until 1926, when James B. Sumner showed that the enzyme urease was in fact a protein.[] The difficulty in purifying proteins in large quantities made them very difficult for early protein biochemists to study. Hence, early studies focused on proteins that could be purified in large quantities, e.g., those of blood, egg white, various toxins, and digestive/metabolic enzymes obtained from slaughterhouses. In the 1950s, the Armour Hot Dog Co. purified 1kg of pure bovine pancreatic ribonuclease A and made it freely available to scientists; this gesture helped ribonuclease A become a major target for biochemical study for the following decades.[] Linus Pauling is credited with the successful prediction of regular protein secondary structures based on hydrogen bonding, an idea first put forth by William Astbury in 1933.[] Later work by Walter Kauzmann on denaturation,[][] based partly on previous studies by Kaj Linderstrøm-Lang,[] contributed an understanding of protein folding and structure mediated by hydrophobic interactions. The first protein to be sequenced was insulin, by Frederick Sanger, in 1949. Sanger correctly determined the amino acid sequence of insulin, thus conclusively demonstrating that proteins consisted of linear polymers of amino acids rather than branched chains, colloids, or cyclols.[] He won the Nobel Prize for this achievement in 1958.

John Kendrew with model of myoglobin in progress.

The first protein structures to be solved were hemoglobin and myoglobin, by Max Perutz and Sir John Cowdery Kendrew, respectively, in 1958.[][] The first atomic-resolution structures of proteins were solved by X-ray diffraction analysis in the 1960s[citation needed] (Perutz and Kendrew shared the 1962 Nobel Prize in Chemistry for these discoveries) and by NMR in the 1980s.[citation needed] As of 2009[20], the Protein Data Bank has over 55,000 atomic-resolution structures of proteins.[] In more recent times, cryo-electron microscopy of large macromolecular

Protein

assemblies[] and computational protein structure prediction of small protein domains[] are two methods approaching atomic resolution.

Footnotes [1] Murray et al., p. 19. [2] Murray et al., p. 31. [3] van Holde and Mathews, pp. 1002–42. [4] Murray et al., p. 36. [5] Murray et al., p. 37. [6] Murray et al., pp. 30–34. [7] van Holde and Mathews, pp. 368–75. [8] van Holde and Mathews, pp. 165–85. [9] Branden and Tooze, pp. 340–41. [10] Voet D, Voet JG. (2004). Biochemistry Vol 1 3rd ed. Wiley: Hoboken, NJ. [11] van Holde and Mathews, pp. 830–49. [12] Branden and Tooze, pp. 251–81. [13] van Holde and Mathews, pp. 247–50. [14] van Holde and Mathews, pp. 220–29. [15] Branden and Tooze, pp. 232–34. [16] van Holde and Mathews, pp. 178–81. [17] [18] [19] [20]

van Holde and Mathews, pp. 258–64; 272. Murray et al., pp. 21–24. New Oxford Dictionary of English http:/ / en. wikipedia. org/ w/ index. php?title=Protein& action=edit

References • Branden C, Tooze J (1999). Introduction to Protein Structure. New York: Garland Pub. ISBN0-8153-2305-0. • Murray RF, Harper HW, Granner DK, Mayes PA, Rodwell VW (2006). Harper's Illustrated Biochemistry. New York: Lange Medical Books/McGraw-Hill. ISBN0-07-146197-3. • Van Holde KE, Mathews CK (1996). Biochemistry. Menlo Park, California: Benjamin/Cummings Pub. Co., Inc. ISBN0-8053-3931-0.

External links Databases and projects • • • • • • • •

The Protein Naming Utility (http://www.jcvi.org/pn-utility) Human Protein Atlas (http://www.proteinatlas.org/) NCBI Entrez Protein database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=protein) NCBI Protein Structure database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=structure) Human Protein Reference Database (http://www.hprd.org/) Human Proteinpedia (http://www.humanproteinpedia.org/) Folding@Home (Stanford University) (http://folding.stanford.edu/) Comparative Toxicogenomics Database (http://ctd.mdibl.org/) curates protein–chemical interactions, as well as gene/protein–disease relationships and chemical-disease relationships. • Bioinformatic Harvester (http://harvester.fzk.de/) A Meta search engine (29 databases) for gene and protein information. • Protein Databank in Europe (http://www.pdbe.org/) (see also PDBeQuips (http://www.pdbe.org/quips), short articles and tutorials on interesting PDB structures) • Research Collaboratory for Structural Bioinformatics (http://www.rcsb.org/) (see also Molecule of the Month (http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/index.html),

Protein presenting short accounts on selected proteins from the PDB) • Proteopedia– Life in 3D (http://www.proteopedia.org/): rotatable, zoomable 3D model with wiki annotations for every known protein molecular structure. • UniProt the Universal Protein Resource (http://www.expasy.uniprot.org/) • neXtProt– Exploring the universe of human proteins (http://www.nextprot.org/): human-centric protein knowledge resource

Tutorials and educational websites • "An Introduction to Proteins" (http://hopes.stanford.edu/basics/proteins/p0.html) from HOPES (Huntington's Disease Outreach Project for Education at Stanford) • Proteins: Biogenesis to Degradation– The Virtual Library of Biochemistry and Cell Biology (http://www. biochemweb.org/proteins.shtml)

Amino acid Amino acids (pron.: /əˈmiːnoʊ/, /əˈmaɪnoʊ/, or /ˈæmɪnoʊ/) are biologically important organic compounds made from amine (-NH2) and carboxylic acid (-COOH) functional groups, along with a side-chain specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, though other elements are found in the side-chains of certain amino acids. About 500 amino acids are known[1] and can be classified in many ways. Structurally they can be classified according to the functional groups' locations as alpha- (α-), beta- (β-), gamma- (γ-) or delta- (δ-) amino acids; other categories relate to polarity, pH The generic structure of an alpha amino acid in its un-ionized form level, and side chain group type (aliphatic, acyclic, aromatic, containing hydroxyl or sulphur, et al.). In the form of proteins, amino acids comprise the second largest component (after water) of human muscles, cells and other tissues.[2] Outside proteins, amino acids perform critical roles in processes such as neurotransmitter transport and biosynthesis. Amino acids having both the amine and carboxylic acid groups attached to the first (alpha-) carbon atom have particular importance in biochemistry. They are known as 2-, alpha-, or α-amino acids (generic formula H2NCHRCOOH in most cases[3] where R is an

Amino acid organic substituent known as a "side-chain");[4] often the term "amino acid" is used to refer specifically to these. They include the 23 proteinogenic ("protein-building") amino acids which combine into peptide chains ("polypeptides") to form the building blocks of a vast array of proteins.[] These are all L-stereoisomers ("left-handed" isomers) although a few D-amino acids ("right-handed") occur in bacterial envelopes and some antibiotics.[5][6] 20 of the 23 proteinogenic amino acids are encoded directly by triplet codons in the genetic code and are known as "standard" amino acids. The other three ("non-standard" or "non-canonical") are pyrrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many noneukaryotes as well as most eukaryotes), and N-Formylmethionine. For example, 25 human proteins include selenocysteine (Sec) in their primary structure,[7] and the structurally characterized enzymes (selenoenzymes) employ Sec as the The 21 amino acids found in eukaryotes, grouped according to their catalytic moiety in their active sites.[8] side-chains' pKa values and charges carried at physiological pH 7.4 Pyrollysine and selenocysteine are encoded via variant codons; for example, selenocysteine is encoded by stop codon and SECIS element.[9][10][] Codon–tRNA combinations not found in nature can also be used to "expand" the genetic code and create novel proteins known as alloproteins incorporating non-proteinogenic amino acids.[][][] Many important proteinogenic and non-proteinogenic amino acids also play critical non-protein roles within the body. For example: in the human brain, glutamate (standard glutamic acid) and gamma-amino-butyric acid ("GABA", non-standard gamma-amino acid) are respectively the main excitatory and inhibitory neurotransmitters;[] hydroxyproline (a major component of the connective tissue collagen) is synthesised from proline; the standard amino acid glycine is used to synthesise porphyrins used in red blood cells; and the non-standard carnitine is used in lipid transport. 9 of the 20 standard amino acids are called "essential" for humans because they cannot be created from other compounds by the human body, and so must be taken in as food. Others may be conditionally essential for certain ages or medical conditions. Essential amino acids may also differ between species.[11] Because of their biological significance, amino acids are important in nutrition and are commonly used in nutritional supplements, fertilizers, and food technology. Industrial uses include the production of drugs, biodegradable plastics and chiral catalysts.

Amino acid

History The first few amino acids were discovered in the early 19th century. In 1806, French chemists Louis-Nicolas Vauquelin and Pierre Jean Robiquet isolated a compound in asparagus that was subsequently named asparagine, the first amino acid to be discovered.[12][] Cystine was discovered in 1810,[13] although its monomer, cysteine, remained undiscovered until 1884.[][14] Glycine and leucine were discovered in 1820.[15] Usage of the term amino acid in the English language is from 1898.[16] Proteins were found to yield amino acids after enzymatic digestion or acid hydrolysis. In 1902, Emil Fischer and Franz Hofmeister proposed that proteins are the result of the formation of bonds between the amino group of one amino acid with the carboxyl group of another, in a linear structure which Fischer termed peptide.[17]

General structure In the structure shown at the top of the page, R represents a side-chain specific to each amino acid. The carbon atom next to the carboxyl group is called the α–carbon and amino acids with a side-chain bonded to this carbon are referred to as alpha amino acids. These are the most common form found in nature. In the alpha amino acids, the α–carbon is a chiral carbon atom, with the exception of glycine.[] In amino acids that have a carbon chain attached to the α–carbon (such as lysine, shown to the right) the carbons are labeled in order as α, β, γ, δ, and so on.[18] In some amino acids, the amine group is attached to the β or γ-carbon, and these are therefore referred to as beta or gamma amino acids. Amino acids are usually classified by the properties Lysine with the carbon atoms in the side-chain labeled of their side-chain into four groups. The side-chain can make an amino acid a weak acid or a weak base, and a hydrophile if the side-chain is polar or a hydrophobe if it is nonpolar.[] The chemical structures of the 22 standard amino acids, along with their chemical properties, are described more fully in the article on these proteinogenic amino acids. The phrase "branched-chain amino acids" or BCAA refers to the amino acids having aliphatic side-chains that are non-linear; these are leucine, isoleucine, and valine. Proline is the only proteinogenic amino acid whose side-group links to the α-amino group and, thus, is also the only proteinogenic amino acid containing a secondary amine at this position.[] In chemical terms, proline is, therefore, an imino acid, since it lacks a primary amino group,[19] although it is still classed as an amino acid in the current biochemical nomenclature,[20] and may also be called an "N-alkylated alpha-amino acid".[21]

Amino acid

Isomerism Of the standard α-amino acids, all but glycine can exist in either of two enantiomers, called L or D amino acids, which are mirror images of each other (see also Chirality). While L-amino acids represent all of the amino acids found in proteins during translation in the ribosome, D-amino acids are found in some proteins produced by enzyme posttranslational modifications after translation and translocation to the endoplasmic reticulum, as in exotic sea-dwelling organisms such as The two enantiomers of alanine, D-Alanine and cone snails.[22] They are also abundant components of the L-Alanine peptidoglycan cell walls of bacteria,[23] and D-serine may act as a neurotransmitter in the brain.[24] The L and D convention for amino acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the isomer of glyceraldehyde from which that amino acid can, in theory, be synthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde is levorotatory). In alternative fashion, the (S) and (R) designators are used to indicate the absolute stereochemistry. Almost all of the amino acids in proteins are (S) at the α carbon, with cysteine being (R) and glycine non-chiral.[25] Cysteine is unusual since it has a sulfur atom at the second position in its side-chain, which has a larger atomic mass than the groups attached to the first carbon, which is attached to the α-carbon in the other standard amino acids, thus the (R) instead of (S).

Zwitterions The amine and carboxylic acid functional groups found in amino acids allow them to have amphiprotic properties.[] Carboxylic acid groups (−CO2H) can be deprotonated to become negative carboxylates (−CO2− ), and α-amino groups (NH2−) can be protonated to become positive + α-ammonium groups ( NH3−). At pH An amino acid in its (1) un-ionized and (2) zwitterionic forms values greater than the pKa of the carboxylic acid group (mean for the 20 common amino acids is about 2.2, see the table of amino acid structures above), the negative carboxylate ion predominates. At pH values lower than the pKa of the α-ammonium group (mean for the 20 common α-amino acids is about 9.4), the nitrogen is predominantly protonated as a positively charged α-ammonium group. Thus, at pH between 2.2 and 9.4, the predominant form adopted by α-amino acids contains a negative carboxylate and a positive α-ammonium group, as shown in structure (2) on the right, so has net zero charge. This molecular state is known as a zwitterion, from the German Zwitter meaning hermaphrodite or hybrid.[26] Below pH 2.2, the predominant form will have a neutral carboxylic acid group and a positive α-ammonium ion (net charge +1), and above pH 9.4, a negative carboxylate and neutral α-amino group (net charge −1). The fully neutral form (structure (1) on the right) is a very minor species in aqueous solution throughout the pH range (less than 1 part in 107). Amino acids also exist as zwitterions in the solid phase, and crystallize with salt-like properties unlike typical organic acids or amines.

Amino acid

Isoelectric point At pH values between the two pKa values, the zwitterion predominates, but coexists in dynamic equilibrium with small amounts of net negative and net positive ions. At the exact midpoint between the two pKa values, the trace amount of net negative and trace of net positive ions exactly balance, so that average net charge of all forms present is zero.[27] This pH is known as the isoelectric point pI, so pI = ½(pKa1 + pKa2). The individual amino acids all have slightly different pKa values, so have different isoelectric points. For amino acids with charged side-chains, the pKa of the side-chain is involved. Thus for Asp, Glu with negative side-chains, pI = ½(pKa1 + pKaR), where pKaR is the side-chain pKa. Cysteine also has potentially negative side-chain with pKaR = 8.14, so pI should be calculated as for Asp and Glu, even though the side-chain is not significantly charged at neutral pH. For His, Lys, and Arg with positive side-chains, pI = ½(pKaR + pKa2). Amino acids have zero mobility in electrophoresis at their isoelectric point, although this behaviour is more usually exploited for peptides and proteins than single amino acids. Zwitterions have minimum solubility at their isolectric point and some amino acids (in particular, with non-polar side-chains) can be isolated by precipitation from water by adjusting the pH to the required isoelectric point.

Occurrence and functions in biochemistry Standard amino acids Amino acids are the structural units (monomers) that make up proteins. They join together to form short polymer chains called peptides or longer chains called either polypeptides or proteins. These polymers are linear and unbranched, with each amino acid within the chain attached to two neighboring amino acids. The process of making proteins is called translation and involves the step-by-step addition of amino A polypeptide is an unbranched chain of amino acids. acids to a growing protein chain by a [28] ribozyme that is called a ribosome. The order in which the amino acids are added is read through the genetic code from an mRNA template, which is a RNA copy of one of the organism's genes. Twenty-two amino acids are naturally incorporated into polypeptides and are called proteinogenic or natural amino acids.[] Of these, 20 are encoded by the universal genetic code. The remaining 2, selenocysteine and pyrrolysine, are incorporated into proteins by unique synthetic mechanisms. Selenocysteine is incorporated when the mRNA being translated includes a SECIS element, which causes the UGA codon to encode selenocysteine instead of a stop codon.[29] Pyrrolysine is used by some methanogenic archaea in enzymes that they use to produce methane. It is coded for with the codon UAG, which is normally a stop codon in other organisms.[30] This UAG codon is followed by a PYLIS downstream sequence.[]

Amino acid

Non-standard amino acids Aside from the 22 standard amino acids, there are many other amino acids that are called non-proteinogenic or non-standard. Those either are not found in proteins (for example carnitine, GABA), or are not produced directly and in isolation by standard cellular machinery (for example, hydroxyproline and selenomethionine). Non-standard amino acids that are found in proteins are formed by post-translational modification, which is modification after translation during protein synthesis. These modifications are often essential for the function or regulation of a protein; for example, the carboxylation of glutamate allows for better binding of calcium cations,[31] and the hydroxylation of proline is critical for maintaining connective tissues.[32] Another example is the formation of hypusine in the translation initiation factor EIF5A, through modification of a lysine residue.[33] Such modifications can also determine the localization of the protein, e.g., the addition of long hydrophobic groups can cause a protein to bind to a phospholipid membrane.[34] The amino acid selenocysteine

Some nonstandard amino acids are not found in proteins. Examples include lanthionine, 2-aminoisobutyric acid, dehydroalanine, and the neurotransmitter gamma-aminobutyric acid. Nonstandard amino acids often occur as intermediates in the metabolic pathways for standard amino acids— for example, ornithine and citrulline occur in the urea cycle, part of amino acid catabolism (see below).[35] A β-alanine and its α-alanine isomer rare exception to the dominance of α-amino acids in biology is the β-amino acid beta alanine (3-aminopropanoic acid), which is used in plants and microorganisms in the synthesis of pantothenic acid (vitamin B5), a component of coenzyme A.[36]

In human nutrition When taken up into the human body from the diet, the 22 standard amino acids either are used to synthesize proteins and other biomolecules or are oxidized to urea and carbon dioxide as a source of energy.[37] The oxidation pathway starts with the removal of the amino group by a transaminase, the amino group is then fed into the urea cycle. The other product of transamidation is a keto acid that enters the citric acid cycle.[38] Glucogenic amino acids can also be converted into glucose, through gluconeogenesis.[39]

Amino acid

Pyrrolysine trait is restricted to several microbes, and only one organism has both Pyl and Sec. Of the 22 standard amino acids, 9 are called essential amino acids because the human body cannot synthesize them from other compounds at the level needed for normal growth, so they must be obtained from food.[40] In addition, cysteine, taurine, tyrosine, and arginine are considered semiessential amino-acids in children (though taurine is not technically an amino acid), because the metabolic pathways that synthesize these amino acids are not fully developed.[41][42] The amounts required also depend on the age and health of the individual, so it is hard to make general statements about the dietary requirement for some amino acids. Essential

Nonessential

Histidine

Alanine

Isoleucine

Arginine*

Leucine

Asparagine

Lysine

Aspartic acid

Methionine

Cysteine*

Phenylalanine Glutamic acid Threonine

Glutamine*

Tryptophan

Glycine

Valine

Ornithine* Proline* Serine* Tyrosine*

(*) Essential only in certain cases.[43][44]

Classification of Amino Acids Although there are many ways to classify amino acids, these molecules can be assorted into six main groups, on the basis of their structure and the general chemical characteristics of their R groups. Class Aliphatic

Name of the amino acids Glycine, Alanine, Valine, Leucine, Isoleucine

Hydroxyl or Sulfur-containing Serine, Cysteine, Threonine, Methionine Cyclic

Proline

Aromatic

Phenylalanine, Tyrosine, Tryptophan

Basic

Histidine, Lysine, Arginine

Acidic and their Amide

Aspartate, Glutamate, Asparagine, Glutamine

Amino acid

Non-protein functions In humans, non-protein amino acids also have important roles as metabolic intermediates, such as in the biosynthesis of the neurotransmitter gamma-aminobutyric acid. Many amino acids are used to synthesize other molecules, for example: • Tryptophan is a precursor of the neurotransmitter serotonin.[45] • Tyrosine (and its precursor phenylalanine) are precursors of the catecholamine neurotransmitters dopamine, epinephrine and norepinephrine. • Glycine is a precursor of porphyrins such as heme.[46] • Arginine is a precursor of nitric oxide.[47] • Ornithine and S-adenosylmethionine are precursors of polyamines.[48] • Aspartate, glycine, and glutamine are precursors of nucleotides.[49] • Phenylalanine is a precursor of various phenylpropanoids, which are important in plant metabolism. However, not all of the functions of other abundant non-standard amino acids are known. Some non-standard amino acids are used as defenses against herbivores in plants.[] For example canavanine is an analogue of arginine that is found in many legumes,[] and in particularly large amounts in Canavalia gladiata (sword bean).[50] This amino acid protects the plants from predators such as insects and can cause illness in people if some types of legumes are eaten without processing.[51] The non-protein amino acid mimosine is found in other species of legume, particularly Leucaena leucocephala.[52] This compound is an analogue of tyrosine and can poison animals that graze on these plants.

Uses in technology Amino acids are used for a variety of applications in industry, but their main use is as additives to animal feed. This is necessary, since many of the bulk components of these feeds, such as soybeans, either have low levels or lack some of the essential amino acids: Lysine, methionine, threonine, and tryptophan are most important in the production of these feeds.[] In this industry, amino acids are also used to chelate metal cations in order to improve the absorption of minerals from supplements, which may be required to improve the health or production of these animals.[53] The food industry is also a major consumer of amino acids, in particular, glutamic acid, which is used as a flavor enhancer,[] and Aspartame (aspartyl-phenylalanine-1-methyl ester) as a low-calorie artificial sweetener.[54] Similar technology to that used for animal nutrition is employed in the human nutrition industry to alleviate symptoms of mineral deficiencies, such as anemia, by improving mineral absorption and reducing negative side effects from inorganic mineral supplementation.[55] The chelating ability of amino acids has been used in fertilizers for agriculture to facilitate the delivery of minerals to plants in order to correct mineral deficiencies, such as iron chlorosis. These fertilizers are also used to prevent deficiencies from occurring and improving the overall health of the plants.[56] The remaining production of amino acids is used in the synthesis of drugs and cosmetics.[]

Amino acid

Amino acid derivative

Pharmaceutical application [57]

5-HTP (5-hydroxytryptophan)

Experimental treatment for depression.

L-DOPA (L-dihydroxyphenylalanine)

Treatment for Parkinsonism.

Eflornithine

Drug that inhibits ornithine decarboxylase and is used in the treatment of sleeping sickness.

[58] [59]

Expanded genetic code Since 2001, 40 non-natural amino acids have been added into protein by creating a unique codon (recoding) and a corresponding transfer-RNA:aminoacyl– tRNA-synthetase pair to encode it with diverse physicochemical and biological properties in order to be used as a tool to exploring protein structure and function or to create novel or enhanced proteins.[][]

Chemical building blocks Amino acids are important as low-cost feedstocks. These compounds are used in chiral pool synthesis as enantiomerically pure building blocks.[] Amino acids have been investigated as precursors chiral catalysts, e.g., for asymmetric hydrogenation reactions, although no commercial applications exist.[]

Biodegradable plastics Amino acids are under development as components of a range of biodegradable polymers. These materials have applications as environmentally friendly packaging and in medicine in drug delivery and the construction of prosthetic implants. These polymers include polypeptides, polyamides, polyesters, polysulfides, and polyurethanes with amino acids either forming part of their main chains or bonded as side-chains. These modifications alter the physical properties and reactivities of the polymers.[] An interesting example of such materials is polyaspartate, a water-soluble biodegradable polymer that may have applications in disposable diapers and agriculture.[] Due to its solubility and ability to chelate metal ions, polyaspartate is also being used as a biodegradeable anti-scaling agent and a corrosion inhibitor.[60][] In addition, the aromatic amino acid tyrosine is being developed as a possible replacement for toxic phenols such as bisphenol A in the manufacture of polycarbonates.[]

Reactions As amino acids have both a primary amine group and a primary carboxyl group, these chemicals can undergo most of the reactions associated with these functional groups. These include nucleophilic addition, amide bond formation and imine formation for the amine group and esterification, amide bond formation and decarboxylation for the carboxylic acid group.[61] The combination of these functional groups allow amino acids to be effective polydentate ligands for metal-amino acid chelates.[62] The multiple side-chains of amino acids can also undergo chemical reactions.[63] The types of these reactions are determined by the groups on these side-chains and are, therefore, different between the various types of amino acid.

Amino acid

Chemical synthesis Several methods exist to synthesize amino acids. One of the oldest methods begins with the bromination at the α-carbon of a carboxylic acid. The Strecker amino acid synthesis Nucleophilic substitution with ammonia then converts the alkyl bromide to the amino acid.[64] In alternative fashion, the Strecker amino acid synthesis involves the treatment of an aldehyde with potassium cyanide and ammonia, this produces an α-amino nitrile as an intermediate. Hydrolysis of the nitrile in acid then yields a α-amino acid.[65] Using ammonia or ammonium salts in this reaction gives unsubstituted amino acids, while substituting primary and secondary amines will yield substituted amino acids.[66] Likewise, using ketones, instead of aldehydes, gives α,α-disubstituted amino [67] acids. The classical synthesis gives racemic mixtures of α-amino acids as products, but several alternative procedures using asymmetric auxiliaries [68] or asymmetric catalysts [69][70] have been developed.[71] At the current time, the most-adopted method is an automated synthesis on a solid support (e.g., polystyrene beads), using protecting groups (e.g., Fmoc and t-Boc) and activating groups (e.g., DCC and DIC).

Peptide bond formation As both the amine and carboxylic acid groups of amino acids can react to form amide bonds, one amino acid molecule can react with another and become joined through an amide linkage. This polymerization of amino acids is what creates proteins. This condensation reaction yields the newly formed peptide bond and a molecule of water. In cells, this reaction does not occur directly; instead the amino acid is first activated by attachment to a transfer RNA molecule through an ester bond. This aminoacyl-tRNA is produced in an ATP-dependent reaction carried out by an aminoacyl tRNA synthetase.[72] This The condensation of two amino acids to form a dipeptide through a peptide bond aminoacyl-tRNA is then a substrate for the ribosome, which catalyzes the attack of the amino group of the elongating protein chain on the ester bond.[73] As a result of this mechanism, all proteins made by ribosomes are synthesized starting at their N-terminus and moving towards their C-terminus. However, not all peptide bonds are formed in this way. In a few cases, peptides are synthesized by specific enzymes. For example, the tripeptide glutathione is an essential part of the defenses of cells against oxidative stress. This peptide is synthesized in two steps from free amino acids.[74] In the first step gamma-glutamylcysteine synthetase condenses cysteine and glutamic acid through a peptide bond formed between the side-chain carboxyl of the glutamate (the gamma carbon of this side-chain) and the amino group of the cysteine. This dipeptide is then condensed with glycine by glutathione synthetase to form glutathione.[75]

Amino acid

In chemistry, peptides are synthesized by a variety of reactions. One of the most-used in solid-phase peptide synthesis uses the aromatic oxime derivatives of amino acids as activated units. These are added in sequence onto the growing peptide chain, which is attached to a solid resin support.[76] The ability to easily synthesize vast numbers of different peptides by varying the types and order of amino acids (using combinatorial chemistry) has made peptide synthesis particularly important in creating libraries of peptides for use in drug discovery through high-throughput screening.[77]

Biosynthesis In plants, nitrogen is first assimilated into organic compounds in the form of glutamate, formed from alpha-ketoglutarate and ammonia in the mitochondrion. In order to form other amino acids, the plant uses transaminases to move the amino group to another alpha-keto carboxylic acid. For example, aspartate aminotransferase converts glutamate and oxaloacetate to alpha-ketoglutarate and aspartate.[78] Other organisms use transaminases for amino acid synthesis, too. Nonstandard amino acids are usually formed through modifications to standard amino acids. For example, homocysteine is formed through the transsulfuration pathway or by the demethylation of methionine via the intermediate metabolite S-adenosyl methionine,[] while hydroxyproline is made by a posttranslational modification of proline.[79] Microorganisms and plants can synthesize many uncommon amino acids. For example, some microbes make 2-aminoisobutyric acid and lanthionine, which is a sulfide-bridged derivative of alanine. Both of these amino acids are found in peptidic lantibiotics such as alamethicin.[80] While in plants, 1-aminocyclopropane-1-carboxylic acid is a small disubstituted cyclic amino acid that is a key intermediate in the production of the plant hormone ethylene.[81]

Catabolism Degradation of an amino acid often involves deamination by moving its amino group to alpha-ketoglutarate, forming glutamate. This process involves transaminases, often the same as those used in amination during synthesis. In many vertebrates, the amino group is then removed through the urea cycle and is excreted in the form of urea. However, amino acid degradation can produce uric acid or ammonia instead. For example, serine dehydratase converts serine to pyruvate and ammonia.[83] After removal of one or more amino groups, the remainder of the molecule can sometimes be used to synthesize new amino acids, or it can be used for energy by entering glycolysis or the citric acid cycle, as detailed in image at right.

Catabolism of proteinogenic amino acids. Amino acids can be classified according [82] to the properties of their main products as either of the following: * Glucogenic, with the products having the ability to form glucose by gluconeogenesis* Ketogenic, with the products not having the ability to form glucose. These products may still be used for ketogenesis or lipid synthesis.* Amino acids catabolized into both glucogenic and ketogenic products.

Physicochemical properties of amino acids

Amino acid

The 20 amino acids encoded directly by the genetic code can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size, and functional groups.[] These properties are important for protein structure and protein–protein interactions. The water-soluble proteins tend to have their hydrophobic residues (Leu, Ile, Val, Phe, and Trp) buried in the middle of the protein, whereas hydrophilic side-chains are exposed to the aqueous solvent. The integral membrane proteins tend to have outer rings of exposed hydrophobic amino acids that anchor them into the lipid bilayer. In the case part-way between these two extremes, some peripheral membrane proteins have a patch of hydrophobic amino acids on their surface that locks onto the membrane. In similar fashion, proteins that have to bind to positively charged molecules have surfaces rich with negatively charged amino acids like glutamate and aspartate, while proteins binding to negatively charged molecules have surfaces rich with positively charged chains like lysine and arginine. There are different hydrophobicity scales of amino acid residues.[84] Some amino acids have special properties such as cysteine, that can form covalent disulfide bonds to other cysteine residues, proline that forms a cycle to the polypeptide backbone, and glycine that is more flexible than other amino acids. Many proteins undergo a range of posttranslational modifications, when additional chemical groups are attached to the amino acids in proteins. Some modifications can produce hydrophobic lipoproteins,[85] or hydrophilic glycoproteins.[86] These type of modification allow the reversible targeting of a protein to a membrane. For example, the addition and removal of the fatty acid palmitic acid to cysteine residues in some signaling proteins causes the proteins to attach and then detach from cell membranes.[87]

Table of standard amino acid abbreviations and properties Amino Acid

[]

3-Letter

[]

1-Letter

Side-chain [] polarity

Side-chain charge [] (pH 7.4)

Hydropathy [88] index

Alanine

Ala

nonpolar

neutral

1.8

Arginine

Arg

Basic polar

positive

−4.5

Asparagine

Asn

polar

neutral

−3.5

Aspartic acid

Asp

acidic polar

negative

−3.5

Cysteine

Cys

nonpolar

neutral

2.5

Glutamic acid

Glu

acidic polar

negative

−3.5

Glutamine

Gln

polar

neutral

−3.5

Glycine

Gly

nonpolar

neutral

−0.4

Further reading • •

• •

Campbell, MK (1999). Biochemistry (Third Edition). Harcourt. ISBN0-03-024426-9 Eshaghian, S; Horwich, TB; Fonarow, GC (January 2006). "An unexpected inverse relationship between HbA1c levels and mortality in patients with diabetes and advanced systolic heart failure". Am Heart J 151 (1): 91. doi: 10.1016/j.ahj.2005.10.008 (http:/ / dx. doi. org/ 10. 1016/ j. ahj. 2005. 10. 008). PMID 16368297 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 16368297). Ganong, WF (2003). Review of Medical Physiology (Twenty-First Edition). Lange. ISBN0-07-140236-5. Hager, T (1995). Force of Nature: The Life of Linus Pauling. Simon and Schuster. ISBN0-684-80909-5.

•

Hardison, RC (June 11, 1996). "A brief history of hemoglobins: plant, animal, protist, and bacteria" (http:/ / www. pubmedcentral. gov/ articlerender. fcgi?tool=pubmed& pubmedid=8650150). Proc Natl Acad Sci USA 93 (12): 5675–9. doi: 10.1073/pnas.93.12.5675 (http:/ / dx. doi. org/ 10. 1073/ pnas. 93. 12. 5675). PMC 39118 (http:/ / www. ncbi. nlm. nih. gov/ pmc/ articles/ PMC39118). PMID 8650150 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 8650150). Kneipp, J; Balakrishnan, G; Chen, R, Shen TJ, Sahu SC, Ho NT, Giovannelli JL, Simplaceanu V, Ho C, Spiro TG; Shen, TJ; Sahu, SC; Ho, NT; Giovannelli, JL; Simplaceanu, V et al. (November 22, 2005). "Dynamics of allostery in hemoglobin: roles of the penultimate tyrosine H bonds". J Mol Biol 356 (2): 335–53. doi: 10.1016/j.jmb.2005.11.006 (http:/ / dx. doi. org/ 10. 1016/ j. jmb. 2005. 11. 006). PMID 16368110 (http:/ / www. ncbi. nlm. nih. gov/ pubmed/ 16368110) . Steinberg, MH (2001). Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management (http:/ / books. google. com/ books?vid=ISBN0521632668). Cambridge University Press. ISBN0-521-63266-8.

External links • Hemoglobin - Test, Levels and Information (http://www.medicinenet.com/hemoglobin/article.htm) on MedicineNet • Interactive hemoglobin saturation curves (http://www.altitude.org/hemoglobin_saturation.php) • Interactive models of hemoglobin (http://www.ufp.pt/~pedros/anim/2frame-hben.htm) (Requires MDL Chime (http://www.mdl.com/products/framework/chime/)) • National Anemia Action Council (http://www.anemia.org/) - anemia.org • New hemoglobin type causes mock diagnosis with pulse oxymeters (http://www.life-of-science.net/medicine/ news/new-hemoglobin-type-discovered-causing-mock-diagnosis-of-cardiac-insufficiency.html)

129

Enzyme mechanisms Enzyme catalysis Enzyme catalysis is the catalysis of chemical reactions by specialized proteins known as enzymes. Catalysis of biochemical reactions in the cell is vital due to the very low reaction rates of the uncatalysed reactions.[citation needed] The mechanism of enzyme catalysis is similar in principle to other types of chemical catalysis. By providing an alternative reaction route the enzyme reduces the energy required to reach the highest energy transition state of the reaction. The reduction of activation energy (Ea) increases the number of reactant molecules with enough energy to reach the activation energy and form the product.

Induced fit The favored model for the enzyme-substrate interaction is the induced fit model.[1] This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding.

Diagrams to show the induced fit hypothesis of enzyme action

The advantages of the induced fit mechanism arise due to the stabilizing effect of strong enzyme binding. There are two different mechanisms of substrate binding: uniform binding, which has strong substrate binding, and differential binding, which has strong transition state binding. The stabilizing effect of uniform binding increases both substrate and transition state binding affinity, while differential binding increases only transition state binding affinity. Both are used by enzymes and have been evolutionarily chosen to minimize the Ea of the reaction. Enzymes which are saturated, that is, have a high affinity substrate binding, require differential binding to reduce the Ea, whereas small substrate unbound enzymes may use either differential or uniform binding.

Enzyme catalysis

130 These effects have led to most proteins using the differential binding mechanism to reduce the Ea, so most proteins have high affinity of the enzyme to the transition state. Differential binding is carried out by the induced fit mechanism - the substrate first binds weakly, then the enzyme changes conformation increasing the affinity to the transition state and stabilizing it, so reducing the activation energy to reach it.

The different mechanisms of substrate binding

It is important to clarify, however, that the induced fit concept cannot be used to rationalize catalysis. That is, the chemical catalysis is defined as the reduction of Ea‡ (when the system is already in the ES‡) relative to Ea‡ in the uncatalyzed reaction in water (without the enzyme). The induced fit only suggests that the barrier is lower in the closed form of the enzyme but does not tell us what the reason for the barrier reduction is. Induced fit may be beneficial to the fidelity of molecular recognition in the presence of competition and noise via the conformational proofreading mechanism .[2]

Mechanisms of an alternative reaction route These conformational changes also bring catalytic residues in the active site close to the chemical bonds in the substrate that will be altered in the reaction. After binding takes place, one or more mechanisms of catalysis lowers the energy of the reaction's transition state, by providing an alternative chemical pathway for the reaction. There are six possible mechanisms of "over the barrier" catalysis as well as a "through the barrier" mechanism:

Bond strain This is the principal effect of induced fit binding, where the affinity of the enzyme to the transition state is greater than to the substrate itself. This induces structural rearrangements which strain substrate bonds into a position closer to the conformation of the transition state, so lowering the energy difference between the substrate and transition state and helping catalyze the reaction. However, the strain effect is, in fact, a ground state destabilization effect, rather than transition state stabilization effect.[3][4] Furthermore, enzymes are very flexible and they cannot apply large strain effect.[5] In addition to bond strain in the substrate, bond strain may also be induced within the enzyme itself to activate residues in the active site.

Enzyme catalysis

131

For example: Substrate, bound substrate, and transition state conformations of lysozyme.

The substrate, on binding, is distorted from the half chair conformation of the hexose ring (because of the steric hindrance with amino acids of the [6] protein forcing the equatorial c6 to be in the axial position) into the chair conformation

Proximity and orientation This increases the rate of the reaction as enzyme-substrate interactions align reactive chemical groups and hold them close together. This reduces the entropy of the reactants and thus makes reactions such as ligations or addition reactions more favorable, there is a reduction in the overall loss of entropy when two reactants become a single product. This effect is analogous to an effective increase in concentration of the reagents. The binding of the reagents to the enzyme gives the reaction intramolecular character, which gives a massive rate increase. For example: Similar reactions will occur far faster if the reaction is intramolecular.

The effective concentration of acetate in the intramolecular reaction can be estimated as k2/k1 = 2 x 105 Molar.

However, the situation might be more complex, since modern computational studies have established that traditional examples of proximity effects cannot be related directly to enzyme entropic effects.[7][8][9] Also, the original entropic proposal[10] has been found to largely overestimate the contribution of orientation entropy to catalysis.[11]

Enzyme catalysis

132

Proton donors or acceptors Proton donors and acceptors, i.e. acids and base may donate and accept protons in order to stabilize developing charges in the transition state.This typically has the effect of activating nucleophile and electrophile groups, or stabilizing leaving groups. Histidine is often the residue involved in these acid/base reactions, since it has a pKa close to neutral pH and can therefore both accept and donate protons. Many reaction mechanisms involving acid/base catalysis assume a substantially altered pKa. This alteration of pKa is possible through the local environment of the residue. Conditions

Acids

Bases

Hydrophobic environment

Increase pKa

Decrease pKa

Adjacent residues of like charge Increase pKa

Decrease pKa

Salt bridge (and hydrogen bond) formation

Decrease pKa Increase pKa

pKa can also be influenced significantly by the surrounding environment, to the extent that residues which are basic in solution may act as proton donors, and vice versa. For example: Serine protease catalytic mechanism

The initial step of the serine protease catalytic mechanism involves the histidine of the active site accepting a proton from the serine residue. This prepares the serine as a nucleophile to attack the amide bond of the substrate. This mechanism includes donation of a proton from serine (a base, pKa 14) to histidine (an acid, pKa 6), made possible due to the local environment of the bases.

It is important to clarify that the modification of the pKa’s is a pure part of the electrostatic mechanism.[4] Furthermore, the catalytic effect of the above example is mainly associated with the reduction of the pKa of the oxyanion and the increase in the pKa of the histidine, while the proton transfer from the serine to the histidine is not catalyzed significantly, since it is not the rate determining barrier.[12]

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Electrostatic catalysis Stabilization of charged transition states can also be by residues in the active site forming ionic bonds (or partial ionic charge interactions) with the intermediate. These bonds can either come from acidic or basic side chains found on amino acids such as lysine, arginine, aspartic acid or glutamic acid or come from metal cofactors such as zinc. Metal ions are particularly effective and can reduce the pKa of water enough to make it an effective nucleophile. Systematic computer simulation studies established that electrostatic effects give, by far, the largest contribution to catalysis.[4] In particular, it has been found that enzyme provides an environment which is more polar than water, and that the ionic transition states are stabilized by fixed dipoles. This is very different from transition state stabilization in water, where the water molecules must pay with "reorganization energy".[13] In order to stabilize ionic and charged states. Thus, the catalysis is associated with the fact that the enzyme polar groups are preorganized [14]

Binding of substrate usually excludes water from the active site, thereby lowering the local dielectric constant to that of an organic solvent. This strengthens the electrostatic interactions between the charged/polar substrates and the active sites. In addition, studies have shown that the charge distributions about the active sites are arranged so as to stabilize the transition states of the catalyzed reactions. In several enzymes, these charge distributions apparently serve to guide polar substrates toward their binding sites so that the rates of these enzymatic reactions are greater than their apparent diffusion-controlled limits. For example: Carboxypeptidase catalytic mechanism

The tetrahedral intermediate is stabilised by a partial ionic bond between the Zn2+ ion and the negative charge on the oxygen.

Covalent catalysis Covalent catalysis involves the substrate forming a transient covalent bond with residues in the active site or with a cofactor. This adds an additional covalent intermediate to the reaction, and helps to reduce the energy of later transition states of the reaction. The covalent bond must, at a later stage in the reaction, be broken to regenerate the enzyme. This mechanism is found in enzymes such as proteases like chymotrypsin and trypsin, where an acyl-enzyme intermediate is formed. Schiff base formation using the free amine from a lysine residue is another mechanism, as seen in the enzyme aldolase during glycolysis. Some enzymes utilize non-amino acid cofactors such as pyridoxal phosphate (PLP) or thiamine pyrophosphate (TPP) to form covalent intermediates with reactant molecules.[15][16] Such covalent intermediates function to reduce the energy of later transition states, similar to how covalent intermediates formed with active site amino acid residues allow stabilization, but the capabilities of cofactors allow enzymes to carryout reactions that amino acid side

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residues alone could not. Enzymes utilizing such cofactors include the PLP-dependent enzyme aspartate transaminase and the TPP-dependent enzyme pyruvate dehydrogenase.[][] It is important to clarify that covalent catalysis does correspond in most cases to simply the use of a specific mechanism rather than to true catalysis.[4] For example, the energetics of the covalent bond to the serine molecule in chymotrypsin should be compared to the well-understood covalent bond to the nucleophile in the uncatalyzed solution reaction. A true proposal of a covalent catalysis (where the barrier is lower than the corresponding barrier in solution) would require, for example, a partial covalent bond to the transition state by an enzyme group (e.g., a very strong hydrogen bond), and such effects do not contribute significantly to catalysis.

Quantum tunneling These traditional "over the barrier" mechanisms have been challenged in some cases by models and observations of "through the barrier" mechanisms (quantum tunneling). Some enzymes operate with kinetics which are faster than what would be predicted by the classical ΔG‡. In "through the barrier" models, a proton or an electron can tunnel through activation barriers.[17][] Quantum tunneling for protons has been observed in tryptamine oxidation by aromatic amine dehydrogenase.[9] Interestingly, quantum tunneling does not appear to provide a major catalytic advantage, since the tunneling contributions are similar in the catalyzed and the uncatalyzed reactions in solution.[][18][19][20] However, the tunneling contribution (typically enhancing rate constants by a factor of ~1000[9] compared to the rate of reaction for the classical 'over the barrier' route) is likely crucial to the viability of biological organisms. This emphasizes the general importance of tunneling reactions in biology. In 1971-1972 the first quantum-mechanical [21][22] formulated. Wikipedia:Independent sources

model

enzyme

catalysis

was

Active enzyme The binding energy of the enzyme-substrate complex cannot be considered as an external energy which is necessary for the substrate activation. The enzyme of high energy content may firstly transfer some specific energetic group X1 from catalytic site of the enzyme to the final place of the first bound reactant, then another group X2 from the second bound reactant (or from the second group of the single reactant) must be transferred to active site to finish substrate conversion to product and enzyme regeneration.[23] We can present the whole enzymatic reaction as a two coupling reactions: S1 + EX1 => S1EX1 => P1 + EP2 S2 + EP2 => S2EP2 => P2 + EX2

(1) (2)

It may be seen from reaction (1) that the group X1 of the active enzyme appears in the product due to possibility of the exchange reaction inside enzyme to avoid both electrostatic inhibition and repulsion of atoms. So we represent the active enzyme as a powerful reactant of the enzymatic reaction. The reaction (2) shows incomplete conversion of the substrate because its group X2 remains inside enzyme. This approach as idea had formerly proposed relying on the hypothetical extremely high enzymatic conversions (catalytically perfect enzyme).[24] The crucial point for the veriﬁcation of the present approach is that the catalyst must be a complex of the enzyme with the transfer group of the reaction. This chemical aspect is supported by the well-studied mechanisms of the several enzymatic reactions. Let us consider the reaction of peptide bond hydrolysis catalyzed by a pure protein α-chymotrypsin (an enzyme acting without a cofactor), which is a well-studied member of the serine proteases family, see.[25] We present the experimental results for this reaction as two chemical steps:

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135

S1 + EH => P1 + EP2 EP2 + H–O–H => EH + P2

(3) (4)

where S1 is a polypeptide, P1 and P2 are products. The first chemical step (3) includes the formation of a covalent acyl-enzyme intermediate. The second step (4) is the deacylation step. It is important to note that the group H+ , initially found on the enzyme, but not in water, appears in the product before the step of hydrolysis, therefore it may be considered as an additional group of the enzymatic reaction. Thus, the reaction (3) shows that the enzyme acts as a powerful reactant of the reaction. According to the proposed concept, the H transport from the enzyme promotes the first reactant conversion, breakdown of the first initial chemical bond (between groups P1 and P2). The step of hydrolysis leads to a breakdown of the second chemical bond and regeneration of the enzyme. The proposed chemical mechanism does not depend on the concentration of the substrates or products in the medium. However, a shift in their concentration mainly causes free energy changes in the first and final steps of the reactions (1) and (2) due to the changes in the free energy content of every molecule, whether S or P, in water solution. This approach is in accordance with the following mechanism of muscle contraction. The final step of ATP hydrolysis in skeletal muscle is the product release caused by the association of myosin heads with actin.[26] The closing of the actin-binding cleft during the association reaction is structurally coupled with the opening of the nucleotide-binding pocket on the myosin active site.[27] Notably, the final steps of ATP hydrolysis include the fast release of phosphate and the slow release of ADP.[28][29] The release of a phosphate anion from bound ADP anion into water solution may be considered as an exergonic reaction because the phosphate anion has low molecular mass. Thus, we arrive at the conclusion that the primary release of the inorganic phosphate H2PO4- leads to transformation of a significant part of the free energy of ATP hydrolysis into the kinetic energy of the solvated phosphate, producing active streaming. This assumption of a local mechano-chemical transduction is in accord with Tirosh’s mechanism of muscle contraction, where the muscle force derives from an integrated action of active streaming created by ATP hydrolysis.[30][31]

Examples of catalytic mechanisms In reality, most enzyme mechanisms involve a combination of several different types of catalysis.

Triose phosphate isomerase Triose phosphate isomerase (EC 5.3.1.1 [32]) catalyses the reversible interconvertion of the two triose phosphates isomers dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate.

Trypsin Trypsin (EC 3.4.21.4 residues.

[33]

) is a serine protease that cleaves protein substrates at lysine and arginine amino acid

Aldolase Aldolase (EC 4.1.2.13 [34]) catalyses the breakdown of fructose 1,6-bisphosphate (F-1,6-BP) into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP).

Enzyme catalysis

References [3] Jencks W.P. "Catalysis in Chemistry and Enzymology" 1987, Dover, New York [4] Warshel A., Sharma P.K., Kato M., Xiang Y., Liu H., and Olsson M.H.M. "Electrostatic Basis of Enzyme Catalysis", Chem. Rev. 2006, 106: 3210-3235. [6] , which is similar in shape to the transition state. [13] Marcus R. A. "On the Theory of Electron-Transfer Reactions. VI. Unified Treatment for Homogeneous and Electrode Reactions" J. Chem. Phys. 1965 43:679-701 [14] Warshel A. "Energetics of Enzyme Catalysis", Proc. Natl. Acad. Sci. USA, 1978 75: 5250 [15] Toney, M. D. "Reaction specificity in pyridoxal enzymes." Archives of biochemistry and biophysics (2005) 433: 279-287 [16] Micronutrient Information Center, Oregon State University (http:/ / lpi. oregonstate. edu/ infocenter/ vitamins/ thiamin/ ) [21] Volkenshtein M.V., Dogonadze R.R., Madumarov A.K., Urushadze Z.D., Kharkats Yu.I. Theory of Enzyme Catalysis.- Molekuliarnaya Biologia, Moscow, 6, 1972, 431-439 [22] Volkenshtein M.V., Dogonadze R.R., Madumarov A.K., Urushadze Z.D., Kharkats Yu.I. Electronic and Conformational Interactions in Enzyme Catalysis. In: E.L. Andronikashvili (Ed.), Konformatsionnie Izmenenia Biopolimerov v Rastvorakh, Publishing House "Nauka", Moscow, 1973, 153-157 [23] Foigel, A.G. (2011). "Is the enzyme a powerful reactant of the biochemical reaction?". Mol. Cell. Biochem 352: 87-89 [24] Fogel, A.G. (1982). "Cooperativity of enzymatic reactions and molecular aspects of energy transduction". Mol. Cell. Biochem 47: 59–64 [25] Hengge, AC, Stein, RL. (2004). "Role of protein conformational mobility in enzyme catalysis: acylation of alpha-chymotrypsin by specific peptide substrates". Biochemistry 43 : 742-747 [26] Lymn, RW, Taylor, EW. (1971). "Mechanism of adenosine triphosphate hydrolysis by actomyosin. Biochemistry 10: 4617–4624 [27] Holmes, KC, Angert, I, Kull, FG, Jahn, W, Schroder, RR. (2003). "Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide". Nature 425: 423–427 [28] Siemankowski, RF, Wiseman, MO, White, HD. (1985). "ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle". Proc. Natl. Acad. Sci. USA 82: 658–662 [29] White, HD, Belknap, B, Webb, MR. (1997). "Kinetics of nucleoside triphosphate cleavage and phosphate release steps by associated rabbit skeletal actomyosin, measured using a novel fluorescent probe for phosphate". Biochemistry 36: 11828–11836 [30] Tirosh, R, Low, WZ, Oplatka, A. (1990). "Translational motion of actin filaments in the presence of heavy meromyosin and MgATP as measured by Doppler broadening of laser light scattering". Biochim. Biophys. Acta 1037: 274–280 [31] Tirosh, R. (2006). "Ballistic protons and microwave-induced water solutions (solitons) in bioenergetic transformations". Int. J. Mol. Sci. 7: 320–345 [32] http:/ / enzyme. expasy. org/ EC/ 5. 3. 1. 1 [33] http:/ / enzyme. expasy. org/ EC/ 3. 4. 21. 4 [34] http:/ / enzyme. expasy. org/ EC/ 4. 1. 2. 13

Further reading • Alan Fersht, Structure and Mechanism in Protein Science : A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman, 1998. ISBN 0-7167-3268-8 • Dedicated issue of Philosophical Transactions B on Quantum catalysis in enzymes freely available. (http:// publishing.royalsociety.org/quantum-catalysis)

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Enzyme kinetics Enzyme kinetics Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction is investigated. Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of this enzyme, its role in metabolism, how its activity is controlled, and how a drug or an agonist might inhibit the enzyme. Enzymes are usually protein molecules that manipulate other molecules — the enzymes' substrates. These target molecules bind to an enzyme's active site and are transformed into products through a series of steps known as the enzymatic mechanism. These mechanisms can be divided into single-substrate and multiple-substrate mechanisms. Kinetic studies on enzymes that only bind one substrate, such as triosephosphate isomerase, aim to measure the affinity with which the enzyme binds this substrate and the turnover rate. Some other examples of enzymes are phosphofructokinase and hexokinase, both of which are important for cellular respiration (glycolysis).

Dihydrofolate reductase from E. coli with its two substrates dihydrofolate (right) and NADPH (left), bound in the active site. The protein is shown as a ribbon diagram, with alpha helices in red, beta sheets in yellow and loops in [1] blue. Generated from 7DFR .

When enzymes bind multiple substrates, such as dihydrofolate reductase (shown right), enzyme kinetics can also show the sequence in which these substrates bind and the sequence in which products are released. An example of enzymes that bind a single substrate and release multiple products are proteases, which cleave one protein substrate into two polypeptide products. Others join two substrates together, such as DNA polymerase linking a nucleotide to DNA. Although these mechanisms are often a complex series of steps, there is typically one rate-determining step that determines the overall kinetics. This rate-determining step may be a chemical reaction or a conformational change of the enzyme or substrates, such as those involved in the release of product(s) from the enzyme. Knowledge of the enzyme's structure is helpful in interpreting kinetic data. For example, the structure can suggest how substrates and products bind during catalysis; what changes occur during the reaction; and even the role of particular amino acid residues in the mechanism. Some enzymes change shape significantly during the mechanism; in such cases, it is helpful to determine the enzyme structure with and without bound substrate analogues that do not undergo the enzymatic reaction.

Enzyme kinetics Not all biological catalysts are protein enzymes; RNA-based catalysts such as ribozymes and ribosomes are essential to many cellular functions, such as RNA splicing and translation. The main difference between ribozymes and enzymes is that RNA catalysts are composed of nucleotides, whereas enzymes are composed of amino acids. Ribozymes also perform a more limited set of reactions, although their reaction mechanisms and kinetics can be analysed and classified by the same methods.

General principles The reaction catalysed by an enzyme uses exactly the same reactants and produces exactly the same products as the uncatalysed reaction. Like other catalysts, enzymes do not alter the position of equilibrium between substrates and products.[2] However, unlike uncatalysed chemical reactions, enzyme-catalysed reactions display saturation kinetics. For a given enzyme concentration and for relatively low substrate concentrations, the reaction rate As larger amounts of substrate are added to a reaction, the available enzyme increases linearly with substrate binding sites become filled to the limit of . Beyond this limit the enzyme is concentration; the enzyme molecules are saturated with substrate and the reaction rate ceases to increase. largely free to catalyse the reaction, and increasing substrate concentration means an increasing rate at which the enzyme and substrate molecules encounter one another. However, at relatively high substrate concentrations, the reaction rate asymptotically approaches the theoretical maximum; the enzyme active sites are almost all occupied and the reaction rate is determined by the intrinsic turnover rate of the enzyme. The substrate concentration midway between these two limiting cases is denoted by KM. The two most important kinetic properties of an enzyme are how quickly the enzyme becomes saturated with a particular substrate, and the maximum rate it can achieve. Knowing these properties suggests what an enzyme might do in the cell and can show how the enzyme will respond to changes in these conditions.

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Enzyme kinetics

Enzyme assays Enzyme assays are laboratory procedures that measure the rate of enzyme reactions. Because enzymes are not consumed by the reactions they catalyse, enzyme assays usually follow changes in the concentration of either substrates or products to measure the rate of reaction. There are many methods of measurement. Spectrophotometric assays observe change in the absorbance of light between products and reactants; radiometric assays involve the incorporation or release of radioactivity to measure the amount of product made over time. Spectrophotometric assays are most convenient since they allow the rate of the reaction to be measured continuously. Although radiometric assays Progress curve for an enzyme reaction. The slope in the initial rate period is the initial rate of reaction v. The Michaelis–Menten require the removal and counting of samples (i.e., they equation describes how this slope varies with the concentration of are discontinuous assays) they are usually extremely substrate. sensitive and can measure very low levels of enzyme activity.[3] An analogous approach is to use mass spectrometry to monitor the incorporation or release of stable isotopes as substrate is converted into product. The most sensitive enzyme assays use lasers focused through a microscope to observe changes in single enzyme molecules as they catalyse their reactions. These measurements either use changes in the fluorescence of cofactors during an enzyme's reaction mechanism, or of fluorescent dyes added onto specific sites of the protein to report movements that occur during catalysis.[4] These studies are providing a new view of the kinetics and dynamics of single enzymes, as opposed to traditional enzyme kinetics, which observes the average behaviour of populations of millions of enzyme molecules.[5][6] An example progress curve for an enzyme assay is shown above. The enzyme produces product at an initial rate that is approximately linear for a short period after the start of the reaction. As the reaction proceeds and substrate is consumed, the rate continuously slows (so long as substrate is not still at saturating levels). To measure the initial (and maximal) rate, enzyme assays are typically carried out while the reaction has progressed only a few percent towards total completion. The length of the initial rate period depends on the assay conditions and can range from milliseconds to hours. However, equipment for rapidly mixing liquids allows fast kinetic measurements on initial rates of less than one second.[7] These very rapid assays are essential for measuring pre-steady-state kinetics, which are discussed below. Most enzyme kinetics studies concentrate on this initial, approximately linear part of enzyme reactions. However, it is also possible to measure the complete reaction curve and fit this data to a non-linear rate equation. This way of measuring enzyme reactions is called progress-curve analysis.[8] This approach is useful as an alternative to rapid kinetics when the initial rate is too fast to measure accurately.

Single-substrate reactions Enzymes with single-substrate mechanisms include isomerases such as triosephosphateisomerase or bisphosphoglycerate mutase, intramolecular lyases such as adenylate cyclase and the hammerhead ribozyme, an RNA lyase.[9] However, some enzymes that only have a single substrate do not fall into this category of mechanisms. Catalase is an example of this, as the enzyme reacts with a first molecule of hydrogen peroxide substrate, becomes oxidised and is then reduced by a second molecule of substrate. Although a single substrate is involved, the existence of a modified enzyme intermediate means that the mechanism of catalase is actually a ping–pong mechanism, a type

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of mechanism that is discussed in the Multi-substrate reactions section below.

Michaelis–Menten kinetics As enzyme-catalysed reactions are saturable, their rate of catalysis does not show a linear response to increasing substrate. If the initial rate of the reaction is measured over a range of substrate concentrations (denoted as [S]), the reaction rate (v) increases as [S] increases, as shown on the right. However, as [S] gets higher, the enzyme becomes saturated with substrate and the rate reaches Vmax, the enzyme's maximum rate. The Michaelis–Menten kinetic model of a single-substrate reaction is shown on the right. There is an initial bimolecular reaction between the enzyme E and substrate S to form the enzyme–substrate complex ES. Although the enzymatic mechanism for the unimolecular reaction can

Saturation curve for an enzyme showing the relation between the concentration of substrate and rate.

be quite complex, there is typically one rate-determining enzymatic step that allows this reaction to be modelled as a single catalytic step with an apparent unimolecular rate constant kcat. If the reaction path Single-substrate mechanism for an enzyme reaction. k1, k−1 and k2 are the rate proceeds over one or several intermediates, constants for the individual steps. kcat will be a function of several elementary rate constants, whereas in the simplest case of a single elementary reaction (e.g. no intermediates) it will be identical to the elementary unimolecular rate constant k2. The apparent unimolecular rate constant kcat is also called turnover number and denotes the maximum number of enzymatic reactions catalysed per second. The Michaelis–Menten equation[10] describes how the (initial) reaction rate v0 depends on the position of the substrate-binding equilibrium and the rate constant k2. (Michaelis–Menten equation) with the constants

This Michaelis–Menten equation is the basis for most single-substrate enzyme kinetics. Two crucial assumptions underlie this equation (apart from the general assumption about the mechanism only involving no intermediate or product inhibition, and there is no allostericity or cooperativity). The first assumption is the so-called quasi-steady-state assumption (or pseudo-steady-state hypothesis), namely that the concentration of the substrate-bound enzyme (and hence also the unbound enzyme) changes much more slowly than those of the product

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and substrate and thus the change over time of the complex can be set to zero

. The second assumption is that th

total enzyme concentration does not change over time, thus

. A complete derivation can be found

The Michaelis constant KM is experimentally defined as the concentration at which the rate of the enzyme reaction is half Vmax, which can be verified by substituting [S] = Km into the Michaelis–Menten equation and can also be seen graphically. If the rate-determining enzymatic step is slow compared to substrate dissociation ( ), the Michaelis constant KM is roughly the dissociation constant KD of the ES complex. If

is small compared to

formed, thus

then the term

and also very little ES complex is

. Therefore, the rate of product formation is

Thus the product formation rate depends on the enzyme concentration as well as on the substrate concentration, the equation resembles a bimolecular reaction with a corresponding pseudo-second order rate constant . This constant is a measure of catalytic efficiency. The most efficient enzymes reach a

in the range of 108 –

1010M−1s−1. These enzymes are so efficient they effectively catalyse a reaction each time they encounter a substrate molecule and have thus reached an upper theoretical limit for efficiency (diffusion limit); these enzymes have often been termed perfect enzymes.[11]

Direct use of the Michaelis–Menten equation for time course kinetic analysis The observed velocities predicted by the Michaelis–Menten equation can be used to directly model the time course disappearance of substrate and the production of product through incorporation of the Michaelis–Menten equation into the equation for first order chemical kinetics. This can only be achieved however if one recognises the problem associated with the use of Euler's number in the description of first order chemical kinetics. i.e. e-k is a split constant that introduces a systematic error into calculations and can be rewritten as a single constant which represents the remaining substrate after each time period.[12]

In 1983 Stuart Beal (and also independently Santiago Schnell and Claudio Mendoza in 1997) derived a closed form solution for the time course kinetics analysis of the Michaelis-Menten mechanism.[13][14] The solution, known as the Schnell-Mendoza equation, has the form:

where W[] is the Lambert-W function.[15][16]

Enzyme kinetics

Linear plots of the Michaelis–Menten equation The plot of v versus [S] above is not linear; although initially linear at low [S], it bends over to saturate at high [S]. Before the modern era of nonlinear curve-fitting on computers, this nonlinearity could make it difficult to estimate KM and Vmax accurately. Therefore, several researchers developed linearisations of the Michaelis–Menten equation, such as the Lineweaver–Burk plot, the Eadie–Hofstee diagram and the Hanes–Woolf plot. All of these linear representations can be useful for Lineweaver–Burk or double-reciprocal plot of kinetic data, showing the significance of visualising data, but none should be the axis intercepts and gradient. used to determine kinetic parameters, as computer software is readily available that allows for more accurate determination by nonlinear regression methods.[17] The Lineweaver–Burk plot or double reciprocal plot is a common way of illustrating kinetic data. This is produced by taking the reciprocal of both sides of the Michaelis–Menten equation. As shown on the right, this is a linear form of the Michaelis–Menten equation and produces a straight line with the equation y = mx + c with a y-intercept equivalent to 1/Vmax and an x-intercept of the graph representing −1/KM.

Naturally, no experimental values can be taken at negative 1/[S]; the lower limiting value 1/[S] = 0 (the y-intercept) corresponds to an infinite substrate concentration, where 1/v=1/Vmax as shown at the right; thus, the x-intercept is an extrapolation of the experimental data taken at positive concentrations. More generally, the Lineweaver–Burk plot skews the importance of measurements taken at low substrate concentrations and, thus, can yield inaccurate estimates of Vmax and KM.[] A more accurate linear plotting method is the Eadie-Hofstee plot. In this case, v is plotted against v/[S]. In the third common linear representation, the Hanes-Woolf plot, [S]/v is plotted against [S]. In general, data normalisation can help diminish the amount of experimental work and can increase the reliability of the output, and is suitable for both graphical and numerical analysis.[18]

Practical significance of kinetic constants The study of enzyme kinetics is important for two basic reasons. Firstly, it helps explain how enzymes work, and secondly, it helps predict how enzymes behave in living organisms. The kinetic constants defined above, KM and Vmax, are critical to attempts to understand how enzymes work together to control metabolism. Making these predictions is not trivial, even for simple systems. For example, oxaloacetate is formed by malate dehydrogenase within the mitochondrion. Oxaloacetate can then be consumed by citrate synthase, phosphoenolpyruvate carboxykinase or aspartate aminotransferase, feeding into the citric acid cycle, gluconeogenesis or aspartic acid biosynthesis, respectively. Being able to predict how much oxaloacetate goes into which pathway requires knowledge of the concentration of oxaloacetate as well as the concentration and kinetics of each of these enzymes. This aim of predicting the behaviour of metabolic pathways reaches its most complex expression in the synthesis of huge amounts of kinetic and gene expression data into mathematical models of entire

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organisms. Alternatively, one useful simplification of the metabolic modelling problem is to ignore the underlying enzyme kinetics and only rely on information about the reaction network's stoichiometry, a technique called flux balance analysis.[19][20]

Michaelis–Menten kinetics with intermediate One could also consider the less simple case

where a complex with the enzyme and an intermediate exists and the intermediate is converted into product in a second step. In this case we have a very similar equation[21]

but the constants are different

We see that for the limiting case

, thus when the last step from EI to E + P is much faster than the

previous step, we get again the original equation. Mathematically we have then

and

Multi-substrate reactions Multi-substrate reactions follow complex rate equations that describe how the substrates bind and in what sequence. The analysis of these reactions is much simpler if the concentration of substrate A is kept constant and substrate B varied. Under these conditions, the enzyme behaves just like a single-substrate enzyme and a plot of v by [S] gives apparent KM and Vmax constants for substrate B. If a set of these measurements is performed at different fixed concentrations of A, these data can be used to work out what the mechanism of the reaction is. For an enzyme that takes two substrates A and B and turns them into two products P and Q, there are two types of mechanism: ternary complex and ping–pong.

Ternary-complex mechanisms In these enzymes, both substrates bind to the enzyme at the same time to produce an EAB ternary complex. The order of binding can either be random (in a random mechanism) or substrates have to bind in a particular sequence (in an ordered mechanism). When a set of v by [S] curves (fixed A, varying B) from an enzyme with a ternary-complex mechanism are plotted in a Lineweaver–Burk plot, the set of lines produced will intersect.

Random-order ternary-complex mechanism for an enzyme reaction. The reaction path is shown as a line and enzyme intermediates containing substrates A and B or products P and Q are written below the line.

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Enzymes with ternary-complex mechanisms include glutathione S-transferase,[22] dihydrofolate reductase[23] and DNA polymerase.[24] The following links show short animations of the ternary-complex mechanisms of the enzymes dihydrofolate reductase[β] and DNA polymerase[γ].

Ping–pong mechanisms As shown on the right, enzymes with a ping-pong mechanism can exist in two states, E and a chemically modified form of the enzyme E*; this modified enzyme Ping–pong mechanism for an enzyme reaction. Intermediates contain substrates A is known as an intermediate. In such and B or products P and Q. mechanisms, substrate A binds, changes the enzyme to E* by, for example, transferring a chemical group to the active site, and is then released. Only after the first substrate is released can substrate B bind and react with the modified enzyme, regenerating the unmodified E form. When a set of v by [S] curves (fixed A, varying B) from an enzyme with a ping–pong mechanism are plotted in a Lineweaver–Burk plot, a set of parallel lines will be produced. This is called a secondary plot. Enzymes with ping–pong mechanisms include some oxidoreductases such as thioredoxin peroxidase,[25] transferases such as acylneuraminate cytidylyltransferase[26] and serine proteases such as trypsin and chymotrypsin.[27] Serine proteases are a very common and diverse family of enzymes, including digestive enzymes (trypsin, chymotrypsin, and elastase), several enzymes of the blood clotting cascade and many others. In these serine proteases, the E* intermediate is an acyl-enzyme species formed by the attack of an active site serine residue on a peptide bond in a protein substrate. A short animation showing the mechanism of chymotrypsin is linked here.[δ]

Non-Michaelis–Menten kinetics

Saturation curve for an enzyme reaction showing sigmoid kinetics.

Some enzymes produce a sigmoid v by [S] plot, which often indicates cooperative binding of substrate to the active site. This means that the binding of one substrate molecule affects the binding of subsequent substrate molecules. This behavior is most common in multimeric enzymes with several interacting active sites.[28] Here, the mechanism of cooperation is similar to that of hemoglobin, with binding of substrate to one active site altering the affinity of the other active sites for substrate molecules. Positive cooperativity occurs when binding of the first substrate molecule increases the affinity of the other active sites for substrate. Negative cooperativity occurs when binding of the first substrate decreases the affinity of

the enzyme for other substrate molecules. Allosteric enzymes include mammalian tyrosyl tRNA-synthetase, which shows negative cooperativity,[29] and bacterial aspartate transcarbamoylase[30] and phosphofructokinase,[31] which show positive cooperativity. Cooperativity is surprisingly common and can help regulate the responses of enzymes to changes in the concentrations of their substrates. Positive cooperativity makes enzymes much more sensitive to [S] and their

Enzyme kinetics activities can show large changes over a narrow range of substrate concentration. Conversely, negative cooperativity makes enzymes insensitive to small changes in [S]. The Hill equation (biochemistry)[32] is often used to describe the degree of cooperativity quantitatively in non-Michaelis–Menten kinetics. The derived Hill coefficient n measures how much the binding of substrate to one active site affects the binding of substrate to the other active sites. A Hill coefficient of 1 indicates positive cooperativity.

Pre-steady-state kinetics In the first moment after an enzyme is mixed with substrate, no product has been formed and no intermediates exist. The study of the next few milliseconds of the reaction is called Pre-steady-state kinetics also referred to as Burst kinetics. Pre-steady-state kinetics is therefore concerned with the formation and consumption of enzyme–substrate intermediates (such as ES or E*) until their steady-state concentrations are reached. This approach was first applied to the hydrolysis reaction catalysed by Pre-steady state progress curve, showing the burst phase of an enzyme reaction. [33] chymotrypsin. Often, the detection of an intermediate is a vital piece of evidence in investigations of what mechanism an enzyme follows. For example, in the ping–pong mechanisms that are shown above, rapid kinetic measurements can follow the release of product P and measure the formation of the modified enzyme intermediate E*.[] In the case of chymotrypsin, this intermediate is formed by an attack on the substrate by the nucleophilic serine in the active site and the formation of the acyl-enzyme intermediate. In the figure to the right, the enzyme produces E* rapidly in the first few seconds of the reaction. The rate then slows as steady state is reached. This rapid burst phase of the reaction measures a single turnover of the enzyme. Consequently, the amount of product released in this burst, shown as the intercept on the y-axis of the graph, also gives the amount of functional enzyme which is present in the assay.[34]

Chemical mechanism An important goal of measuring enzyme kinetics is to determine the chemical mechanism of an enzyme reaction, i.e., the sequence of chemical steps that transform substrate into product. The kinetic approaches discussed above will show at what rates intermediates are formed and inter-converted, but they cannot identify exactly what these intermediates are. Kinetic measurements taken under various solution conditions or on slightly modified enzymes or substrates often shed light on this chemical mechanism, as they reveal the rate-determining step or intermediates in the reaction. For example, the breaking of a covalent bond to a hydrogen atom is a common rate-determining step. Which of the possible hydrogen transfers is rate determining can be shown by measuring the kinetic effects of substituting each hydrogen by deuterium, its stable isotope. The rate will change when the critical hydrogen is replaced, due to a primary kinetic isotope effect, which occurs because bonds to deuterium are harder to break than bonds to hydrogen.[35] It is also possible to measure similar effects with other isotope substitutions, such as 13C/12C and 18 16 O/ O, but these effects are more subtle.[36]

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Isotopes can also be used to reveal the fate of various parts of the substrate molecules in the final products. For example, it is sometimes difficult to discern the origin of an oxygen atom in the final product; since it may have come from water or from part of the substrate. This may be determined by systematically substituting oxygen's stable isotope 18O into the various molecules that participate in the reaction and checking for the isotope in the product.[37] The chemical mechanism can also be elucidated by examining the kinetics and isotope effects under different pH conditions,[38] by altering the metal ions or other bound cofactors,[39] by site-directed mutagenesis of conserved amino acid residues, or by studying the behaviour of the enzyme in the presence of analogues of the substrate(s).[40]

Enzyme inhibition and activation Enzyme inhibitors are molecules that reduce or abolish enzyme activity, while enzyme activators are molecules that increase the catalytic rate of enzymes. These interactions can be either reversible (i.e., removal of the inhibitor restores enzyme activity) or irreversible (i.e., the inhibitor permanently inactivates the enzyme).

Reversible inhibitors

Kinetic scheme for reversible enzyme inhibitors.

Traditionally reversible enzyme inhibitors have been classified as competitive, uncompetitive, or non-competitive, according to their effects on Km and Vmax. These different effects result from the inhibitor binding to the enzyme E, to the enzyme–substrate complex ES, or to both, respectively. The division of these classes arises from a problem in their derivation and results in the need to use two different binding constants for one binding event. The binding of an inhibitor and its effect on the enzymatic activity are two distinctly different things, another problem the traditional equations fail to acknowledge. In noncompetitive inhibition the binding of the inhibitor results in 100% inhibition of the enzyme only, and fails to consider the possibility of anything in between.[41] The common form of the inhibitory term also obscures the relationship between the inhibitor binding to the enzyme and its relationship to any other binding term be it the Michaelis–Menten equation or a dose response curve associated with ligand receptor binding. To demonstrate the relationship the following rearrangement can be made:

Adding zero to the bottom ([I]-[I])

Dividing by [I]+Ki

Enzyme kinetics

This notation demonstrates that similar to the Michaelis–Menten equation,where the rate of reaction depends on the percent of the enzyme population interacting with substrate fraction of the enzyme population bound by substrate

fraction of the enzyme population bound by inhibitor

the effect of the inhibitor is a result of the percent of the enzyme population interacting with inhibitor. The only problem with this equation in its present form is that it assumes absolute inhibition of the enzyme with inhibitor binding, when in fact there can be a wide range of effects anywhere from 100% inhibition of substrate turn over to just >0%. To account for this the equation can be easily modified to allow for different degrees of inhibition by including a delta Vmax term.

This term can then define the residual enzymatic activity present when the inhibitor is interacting with individual enzymes in the population. However the inclusion of this term has the added value of allowing for the possibility of activation if the secondary Vmax term turns out to be higher than the initial term. To account for the possibly of activation as well the notation can then be rewritten replacing the inhibitor "I" with a modifier term denoted here as "X".

While this terminology results in a simplified way of dealing with kinetic effects relating to the maximum velocity of the Michaelis–Menten equation, it highlights potential problems with the term used to describe effects relating to the Km. The Km relating to the affinity of the enzyme for the substrate should in most cases relate to potential changes in the binding site of the enzyme which would directly result from enzyme inhibitor interactions. As such a term similar to the one proposed above to modulate Vmax should be appropriate in most situations:[42][43]

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Irreversible inhibitors Enzyme inhibitors can also irreversibly inactivate enzymes, usually by covalently modifying active site residues. These reactions, which may be called suicide substrates, follow exponential decay functions and are usually saturable. Below saturation, they follow first order kinetics with respect to inhibitor.

Mechanisms of catalysis The favoured model for the enzyme–substrate interaction is the induced fit model.[44] This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding. These conformational changes also bring catalytic residues in the active site close to the chemical bonds in the substrate that will be altered in the reaction.[45] Conformational changes can be measured using circular dichroism or dual polarisation interferometry. After binding takes place, one or more mechanisms of The energy variation as a function of reaction coordinate shows the stabilisation of catalysis lower the energy of the reaction's the transition state by an enzyme. transition state by providing an alternative chemical pathway for the reaction. Mechanisms of catalysis include catalysis by bond strain; by proximity and orientation; by active-site proton donors or acceptors; covalent catalysis and quantum tunnelling.[][46] Enzyme kinetics cannot prove which modes of catalysis are used by an enzyme. However, some kinetic data can suggest possibilities to be examined by other techniques. For example, a ping–pong mechanism with burst-phase pre-steady-state kinetics would suggest covalent catalysis might be important in this enzyme's mechanism. Alternatively, the observation of a strong pH effect on Vmax but not Km might indicate that a residue in the active site needs to be in a particular ionisation state for catalysis to occur.

Software ENZO ENZO (Enzyme Kinetics) is a graphical interface tool for building kinetic models of enzyme catalyzed reactions. ENZO automatically generates the corresponding differential equations from a stipulated enzyme reaction scheme. These differential equations are processed by a numerical solver and a regression algorithm which fits the coefficients of differential equations to experimentally observed time course curves. ENZO allows rapid evaluation of rival reaction schemes and can be used for routine tests in enzyme kinetics.[47]

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Footnotes α. ^ Link: Interactive Michaelis–Menten kinetics tutorial (Java required) [48] β. ^ Link: dihydrofolate reductase mechanism (Gif) [49] γ. ^ Link: DNA polymerase mechanism (Gif) [50] δ. ^ Link: Chymotrypsin mechanism (Flash required) [51]

References [1] http:/ / www. rcsb. org/ pdb/ explore. do?structureId=7DFR [10] Michaelis L. and Menten M.L. Kinetik der Invertinwirkung Biochem. Z. 1913; 49:333–369 English translation (http:/ / web. lemoyne. edu/ ~giunta/ menten. html) Accessed 6 April 2007 [12] Walsh R, Martin E, Darvesh S. A method to describe enzyme-catalyzed reactions by combining steady state and time course enzyme kinetic parameters. Biochim Biophys Acta. 2010 Jan;1800:1–5 [21] for a complete derivation, see here [32] Hill, A. V. The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J. Physiol. (Lond.), 1910 40, iv–vii. [47] ENZO server (http:/ / enzo. cmm. ki. si/ ) [48] http:/ / cti. itc. virginia. edu/ ~cmg/ Demo/ scriptFrame. html [49] http:/ / chem-faculty. ucsd. edu/ kraut/ dhfr. html [50] http:/ / chem-faculty. ucsd. edu/ kraut/ dNTP. html [51] http:/ / web. archive. org/ web/ 20070319235224/ http:/ / courses. cm. utexas. edu/ jrobertus/ ch339k/ overheads-2/ 06_21_chymotrypsin. html

Further reading Introductory • Cornish-Bowden, Athel (2004). Fundamentals of enzyme kinetics (3rd ed.). London: Portland Press. ISBN1-85578-158-1. • Stevens, Lewis; Price, Nicholas C. (1999). Fundamentals of enzymology: the cell and molecular biology of catalytic proteins. Oxford [Oxfordshire]: Oxford University Press. ISBN0-19-850229-X. • Bugg, Tim (2004). Introduction to Enzyme and Coenzyme Chemistry. Cambridge, MA: Blackwell Publishers. ISBN1-4051-1452-5. Advanced • Segel, Irwin H. (1993). Enzyme kinetics: behavior and analysis of rapid equilibrium and steady state enzyme systems (New ed.). New York: Wiley. ISBN0-471-30309-7. • Fersht, Alan (1999). Structure and mechanism in protein science: a guide to enzyme catalysis and protein folding. San Francisco: W.H. Freeman. ISBN0-7167-3268-8. • Santiago Schnell, Philip K. Maini (2004). "A century of enzyme kinetics: Reliability of the KM and vmax estimates" (http://web.archive.org/web/20060221045110/http://www.informatics.indiana.edu/schnell/ papers/ctb8_169.pdf). Comments on Theoretical Biology 8 (2–3): 169–87. doi: 10.1080/08948550302453 (http:/ /dx.doi.org/10.1080/08948550302453). • Walsh, Christopher (1979). Enzymatic reaction mechanisms. San Francisco: W. H. Freeman. ISBN0-7167-0070-0. • Cleland, William Wallace; Cook, Paul (2007). Enzyme kinetics and mechanism. New York: Garland Science. ISBN0-8153-4140-7.

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External links • Animation of an enzyme assay (http://www.kscience.co.uk/animations/model.swf) — Shows effects of manipulating assay conditions • MACiE (http://www.ebi.ac.uk/thornton-srv/databases/MACiE/) — A database of enzyme reaction mechanisms • ENZYME (http://us.expasy.org/enzyme/) — Expasy enzyme nomenclature database • ENZO (http://enzo.cmm.ki.si) — Web application for easy construction and quick testing of kinetic models of enzyme catalyzed reactions. • ExCatDB (http://mbs.cbrc.jp/EzCatDB/) — A database of enzyme catalytic mechanisms • BRENDA (http://www.brenda-enzymes.info/) — Comprehensive enzyme database, giving substrates, inhibitors and reaction diagrams • SABIO-RK (http://sabio.h-its.org) — A database of reaction kinetics • Joseph Kraut's Research Group, University of California San Diego (http://chem-faculty.ucsd.edu/kraut/dhfr. html) — Animations of several enzyme reaction mechanisms • Symbolism and Terminology in Enzyme Kinetics (http://www.chem.qmul.ac.uk/iubmb/kinetics/) — A comprehensive explanation of concepts and terminology in enzyme kinetics • An introduction to enzyme kinetics (http://web.archive.org/web/20040612065857/http://orion1.paisley.ac. uk/kinetics/contents.html) — An accessible set of on-line tutorials on enzyme kinetics • Enzyme kinetics animated tutorial (http://www.wiley.com/college/pratt/0471393878/student/animations/ enzyme_kinetics/index.html) — An animated tutorial with audio

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Lipids and membranes Lipid Lipids constitute a group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, phospholipids, and others. The main biological functions of lipids include energy storage, signaling, and acting as structural components of cell membranes.[][] Lipids have found applications in cosmetic and food industries as well as in nanotechnology.[3] Lipids may be broadly defined as hydrophobic or amphiphilic small molecules; the amphiphilic nature of some lipids allows them to form structures such as vesicles, liposomes, or membranes in an aqueous [] [1] environment. Biological lipids Structures of some common lipids. At the top are cholesterol and oleic acid. The originate entirely or in part from two middle structure is a triglyceride composed of oleoyl, stearoyl, and palmitoyl chains attached to a glycerol backbone. At the bottom is the common phospholipid, distinct types of biochemical subunits [2] phosphatidylcholine. or "building-blocks": ketoacyl and isoprene groups.[] Using this approach, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides (derived from condensation of ketoacyl subunits); and sterol lipids and prenol lipids (derived from condensation of isoprene subunits).[] Although the term lipid is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as other sterol-containing metabolites such as cholesterol.[4] Although humans and other mammals use various biosynthetic pathways to both break down and synthesize lipids, some essential lipids cannot be made this way and must be obtained from the diet.

Lipid

Categories of lipids Fatty acids Fatty acids, or fatty acid residues when they form part of a lipid, are a diverse group of molecules synthesized by chain-elongation of an acetyl-CoA primer with malonyl-CoA or methylmalonyl-CoA groups in a process called fatty acid synthesis.[][] They are made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The fatty acid structure is one of the most fundamental categories of biological lipids, and is commonly used as a building-block of more structurally complex lipids.[5] The carbon chain, typically between four and 24 carbons long,[] may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. Where a double bond exists, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain. This in turn plays an important role in the structure and function of cell membranes.[6] Most naturally occurring fatty acids are of the cis configuration, although the trans form does exist in some natural and partially hydrogenated fats and oils.[] Examples of biologically important fatty acids are the eicosanoids, derived primarily from arachidonic acid and eicosapentaenoic acid, that include prostaglandins, leukotrienes, and thromboxanes. Docosahexaenoic acid is also important in biological systems, particularly with respect to sight.[][7] Other major lipid classes in the fatty acid category are the fatty esters and fatty amides. Fatty esters include important biochemical intermediates such as wax esters, fatty acid thioester coenzyme A derivatives, fatty acid thioester ACP derivatives and fatty acid carnitines. The fatty amides include N-acyl ethanolamines, such as the cannabinoid neurotransmitter anandamide.[]

Glycerolipids Glycerolipids are composed mainly of mono-, di-, and tri-substituted glycerols,[8] the most well-known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride", though the latter lipid contain no hydroxyl group. In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Because they function as an energy store, these lipids comprise the bulk of storage fat in animal tissues. The hydrolysis of the ester bonds of triglycerides and the release of glycerol and fatty acids from adipose tissue are the initial steps in metabolising fat.[9] Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage. Examples of structures in this category are the digalactosyldiacylglycerols found in plant membranes[] and seminolipid from mammalian sperm cells.[]

Glycerophospholipids Glycerophospholipids, usually referred to as phospholipids, are ubiquitous in nature and are key components of the lipid bilayer of cells,[] as well as being involved in metabolism and cell signaling.[] Neural tissue (including the brain) contains relatively high amounts of glycerophospholipids, and alterations in their composition has been implicated in various neurological disorders.[] Glycerophospholipids may be subdivided into distinct classes, based on the nature of the polar headgroup at the sn-3 position of the glycerol backbone in eukaryotes and eubacteria, or the sn-1 position in the case of archaebacteria.[]

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Examples of glycerophospholipids found in biological membranes are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer). In addition to serving as a primary component of cellular membranes and binding sites for intra- and intercellular proteins, some glycerophospholipids in eukaryotic cells, such as phosphatidylinositols Phosphatidylethanolamine and phosphatidic acids are either precursors of or, themselves, membrane-derived second messengers.[10] Typically, one or both of these hydroxyl groups are acylated with long-chain fatty acids, but there are also alkyl-linked and 1Z-alkenyl-linked (plasmalogen) glycerophospholipids, as well as dialkylether variants in archaebacteria.[]

Sphingolipids Sphingolipids are a complicated family of compounds[11] that share a common structural feature, a sphingoid base backbone that is synthesized de novo from the amino acid serine and a long-chain fatty acyl CoA, then converted into ceramides, phosphosphingolipids, glycosphingolipids and other compounds. The major sphingoid base of mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms.[12] The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines),[13] whereas insects contain mainly ceramide phosphoethanolamines[] and fungi have phytoceramide phosphoinositols and mannose-containing headgroups.[] The Sphingomyelin glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.

Sterol lipids Sterol lipids, such as cholesterol and its derivatives, are an important component of membrane lipids,[14] along with the glycerophospholipids and sphingomyelins. The steroids, all derived from the same fused four-ring core structure, have different biological roles as hormones and signaling molecules. The eighteen-carbon (C18) steroids include the estrogen family whereas the C19 steroids comprise the androgens such as testosterone and androsterone. The C21 subclass includes the progestogens as well as the glucocorticoids and mineralocorticoids.[15] The secosteroids, comprising various forms of vitamin D, are characterized by cleavage of the B ring of the core structure.[] Other examples of sterols are the bile acids and their conjugates,[16] which in mammals are oxidized derivatives of cholesterol and are synthesized in the liver. The plant equivalents are the phytosterols, such as β-sitosterol, stigmasterol, and brassicasterol; the latter compound is also used as a biomarker for algal growth.[17] The predominant sterol in fungal cell membranes is ergosterol.[]

Prenol lipids Prenol lipids are synthesized from the five-carbon-unit precursors isopentenyl diphosphate and dimethylallyl diphosphate that are produced mainly via the mevalonic acid (MVA) pathway.[18] The simple isoprenoids (linear alcohols, diphosphates, etc.) are formed by the successive addition of C5 units, and are classified according to number of these terpene units. Structures containing greater than 40 carbons are known as polyterpenes. Carotenoids are important simple isoprenoids that function as antioxidants and as precursors of vitamin A.[] Another biologically important class of molecules is exemplified by the quinones and hydroquinones, which contain an isoprenoid tail attached to a quinonoid core of non-isoprenoid origin.[] Vitamin E and vitamin K, as well as the ubiquinones, are

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examples of this class. Prokaryotes synthesize polyprenols (called bactoprenols) in which the terminal isoprenoid unit attached to oxygen remains unsaturated, whereas in animal polyprenols (dolichols) the terminal isoprenoid is reduced.[]

Saccharolipids Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the LipidA component of the lipopolysaccharides in Gram-negative bacteria. Typical lipidA molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.[]

[] Structure of the saccharolipid Kdo2-Lipid A. Glucosamine residues in blue, Kdo residues in red, acyl chains in black and phosphate groups in green.

Polyketides Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity.[19][] Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, and/or other processes. Many commonly used anti-microbial, anti-parasitic, and anti-cancer agents are polyketides or polyketide derivatives, such as erythromycins, tetracyclines, avermectins, and antitumor epothilones.[]

Biological functions Membranes Eukaryotic cells are compartmentalized into membrane-bound organelles that carry out different biological functions. The glycerophospholipids are the main structural component of biological membranes, such as the cellular plasma membrane and the intracellular membranes of organelles; in animal cells the plasma membrane physically separates the intracellular components from the extracellular environment. The glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. While glycerophospholipids are the major component of biological membranes, other non-glyceride lipid components such

Lipid as sphingomyelin and sterols (mainly cholesterol in animal cell membranes) are also found in biological membranes.[20] In plants and algae, the galactosyldiacylglycerols,[21] and sulfoquinovosyldiacylglycerol,[] which lack a phosphate group, are important components of membranes of chloroplasts and related organelles and are the most abundant lipids in photosynthetic tissues, including those of higher plants, algae and certain bacteria. Bilayers have been found to exhibit high levels of birefringence, which can be used to probe the degree of order (or disruption) within the bilayer using techniques such as dual polarization interferometry and Circular dichroism. A biological membrane is a form of lipid bilayer. The formation of lipid bilayers is an energetically preferred process when the glycerophospholipids described above are in an aqueous environment.[22] This is known as the hydrophobic effect. In an aqueous system, the polar heads of lipids align towards the polar, aqueous environment, while the hydrophobic tails minimize their contact with water and tend to cluster together, forming a vesicle; depending on the concentration of the lipid, this biophysical interaction may result in the formation of micelles, liposomes, or lipid bilayers. Other aggregations are also observed and form part of the polymorphism of amphiphile (lipid) behavior. Phase behavior is an area of study within biophysics and is the subject of current academic research.[][] Micelles and bilayers form in the polar medium by a process known as the hydrophobic effect.[23] When dissolving a lipophilic or amphiphilic substance in a polar environment, the polar molecules (i.e., water in Self-organization of phospholipids: a spherical liposome, a micelle, an aqueous solution) become more ordered around the and a lipid bilayer. dissolved lipophilic substance, since the polar molecules cannot form hydrogen bonds to the lipophilic areas of the amphiphile. So in an aqueous environment, the water molecules form an ordered "clathrate" cage around the dissolved lipophilic molecule.[24]

Energy storage Triglycerides, stored in adipose tissue, are a major form of energy storage both in animals and plants. The adipocyte, or fat cell, is designed for continuous synthesis and breakdown of triglycerides in animals, with breakdown controlled mainly by the activation of hormone-sensitive enzyme lipase.[25] The complete oxidation of fatty acids provides high caloric content, about 9kcal/g, compared with 4kcal/g for the breakdown of carbohydrates and proteins. Migratory birds that must fly long distances without eating use stored energy of triglycerides to fuel their flights.[26]

Signaling In recent years, evidence has emerged showing that lipid signaling is a vital part of the cell signaling.[27][28] Lipid signaling may occur via activation of G protein-coupled or nuclear receptors, and members of several different lipid categories have been identified as signaling molecules and cellular messengers.[29] These include sphingosine-1-phosphate, a sphingolipid derived from ceramide that is a potent messenger molecule involved in regulating calcium mobilization,[] cell growth, and apoptosis;[] diacylglycerol (DAG) and the phosphatidylinositol

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Lipid phosphates (PIPs), involved in calcium-mediated activation of protein kinase C;[] the prostaglandins, which are one type of fatty-acid derived eicosanoid involved in inflammation and immunity;[] the steroid hormones such as estrogen, testosterone and cortisol, which modulate a host of functions such as reproduction, metabolism and blood pressure; and the oxysterols such as 25-hydroxy-cholesterol that are liver X receptor agonists.[] Phosphatidylserine lipids are known to be involved in signaling for the phagocytosis of apoptotic cells and/or pieces of cells. They accomplish this by being exposed to the extracellular face of the cell membrane after the inactivation of flippases which place them exclusively on the cytosolic side and the activation of scramblases, which scramble the orientation of the phospholipids. After this occurs, other cells recognize the phosphatidylserines and phagocytosize the cells or cell fragments exposing them.[citation needed]

Other functions The "fat-soluble" vitamins (A, D, E and K)– which are isoprene-based lipids– are essential nutrients stored in the liver and fatty tissues, with a diverse range of functions. Acyl-carnitines are involved in the transport and metabolism of fatty acids in and out of mitochondria, where they undergo beta oxidation.[30] Polyprenols and their phosphorylated derivatives also play important transport roles, in this case the transport of oligosaccharides across membranes. Polyprenol phosphate sugars and polyprenol diphosphate sugars function in extra-cytoplasmic glycosylation reactions, in extracellular polysaccharide biosynthesis (for instance, peptidoglycan polymerization in bacteria), and in eukaryotic protein N-glycosylation.[31][32] Cardiolipins are a subclass of glycerophospholipids containing four acyl chains and three glycerol groups that are particularly abundant in the inner mitochondrial membrane.[33][34][] They are believed to activate enzymes involved with oxidative phosphorylation.[35] Lipids also form the basis of steroid hormones.[36]

Metabolism The major dietary lipids for humans and other animals are animal and plant triglycerides, sterols, and membrane phospholipids. The process of lipid metabolism synthesizes and degrades the lipid stores and produces the structural and functional lipids characteristic of individual tissues.

Biosynthesis In animals, when there is an oversupply of dietary carbohydrate, the excess carbohydrate is converted to triglycerides. This involves the synthesis of fatty acids from acetyl-CoA and the esterification of fatty acids in the production of triglycerides, a process called lipogenesis.[37] Fatty acids are made by fatty acid synthases that polymerize and then reduce acetyl-CoA units. The acyl chains in the fatty acids are extended by a cycle of reactions that add the acetyl group, reduce it to an alcohol, dehydrate it to an alkene group and then reduce it again to an alkane group. The enzymes of fatty acid biosynthesis are divided into two groups, in animals and fungi all these fatty acid synthase reactions are carried out by a single multifunctional protein,[38] while in plant plastids and bacteria separate enzymes perform each step in the pathway.[39][40] The fatty acids may be subsequently converted to triglycerides that are packaged in lipoproteins and secreted from the liver. The synthesis of unsaturated fatty acids involves a desaturation reaction, whereby a double bond is introduced into the fatty acyl chain. For example, in humans, the desaturation of stearic acid by stearoyl-CoA desaturase-1 produces oleic acid. The doubly unsaturated fatty acid linoleic acid as well as the triply unsaturated α-linolenic acid cannot be synthesized in mammalian tissues, and are therefore essential fatty acids and must be obtained from the diet.[41] Triglyceride synthesis takes place in the endoplasmic reticulum by metabolic pathways in which acyl groups in fatty acyl-CoAs are transferred to the hydroxyl groups of glycerol-3-phosphate and diacylglycerol.[42] Terpenes and isoprenoids, including the carotenoids, are made by the assembly and modification of isoprene units donated from the reactive precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate.[] These precursors can be made in different ways. In animals and archaea, the mevalonate pathway produces these compounds from

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Lipid acetyl-CoA,[43] while in plants and bacteria the non-mevalonate pathway uses pyruvate and glyceraldehyde 3-phosphate as substrates.[][44] One important reaction that uses these activated isoprene donors is steroid biosynthesis. Here, the isoprene units are joined together to make squalene and then folded up and formed into a set of rings to make lanosterol.[] Lanosterol can then be converted into other steroids such as cholesterol and ergosterol.[][45]

Degradation Beta oxidation is the metabolic process by which fatty acids are broken down in the mitochondria and/or in peroxisomes to generate acetyl-CoA. For the most part, fatty acids are oxidized by a mechanism that is similar to, but not identical with, a reversal of the process of fatty acid synthesis. That is, two-carbon fragments are removed sequentially from the carboxyl end of the acid after steps of dehydrogenation, hydration, and oxidation to form a beta-keto acid, which is split by thiolysis. The acetyl-CoA is then ultimately converted into ATP, CO2, and H2O using the citric acid cycle and the electron transport chain. Hence the Krebs Cycle can start at acetyl-CoA when fat is being broken down for energy if there is little or no glucose available. The energy yield of the complete oxidation of the fatty acid palmitate is 106 ATP.[46] Unsaturated and odd-chain fatty acids require additional enzymatic steps for degradation.

Nutrition and health Most of the fat found in food is in the form of triglycerides, cholesterol, and phospholipids. Some dietary fat is necessary to facilitate absorption of fat-soluble vitamins (A, D, E, and K) and carotenoids.[47] Humans and other mammals have a dietary requirement for certain essential fatty acids, such as linoleic acid (an omega-6 fatty acid) and alpha-linolenic acid (an omega-3 fatty acid) because they cannot be synthesized from simple precursors in the diet.[41] Both of these fatty acids are 18-carbon polyunsaturated fatty acids differing in the number and position of the double bonds. Most vegetable oils are rich in linoleic acid (safflower, sunflower, and corn oils). Alpha-linolenic acid is found in the green leaves of plants, and in selected seeds, nuts, and legumes (in particular flax, rapeseed, walnut, and soy).[] Fish oils are particularly rich in the longer-chain omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).[48] A large number of studies have shown positive health benefits associated with consumption of omega-3 fatty acids on infant development, cancer, cardiovascular diseases, and various mental illnesses, such as depression, attention-deficit hyperactivity disorder, and dementia.[][] In contrast, it is now well-established that consumption of trans fats, such as those present in partially hydrogenated vegetable oils, are a risk factor for cardiovascular disease.[][][] A few studies have suggested that total dietary fat intake is linked to an increased risk of obesity[][49] and diabetes.[50][] However, a number of very large studies, including the Women's Health Initiative Dietary Modification Trial, an eight-year study of 49,000 women, the Nurses' Health Study and the Health Professionals Follow-up Study, revealed no such links.[][][] None of these studies suggested any connection between percentage of calories from fat and risk of cancer, heart disease, or weight gain. The Nutrition Source, a website maintained by the Department of Nutrition at the Harvard School of Public Health, summarizes the current evidence on the impact of dietary fat: "Detailed research—much of it done at Harvard—shows that the total amount of fat in the diet isn't really linked with weight or disease."[]

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References [1] Stryer et al., p. 328. [2] Stryer et al., p. 330. [6] Devlin, pp. 193–95. [9] van Holde and Mathews, p. 630–31. [10] van Holde and Mathews, p. 844. [11] Merrill AH, Sandhoff K. (2002). "Sphingolipids: metabolism and cell signaling",in New Comprehensive Biochemistry: Biochemistry of Lipids, Lipoproteins,and Membranes, Vance, D.E. and Vance, J.E., eds. Elsevier Science, NY. Ch. 14. [12] Devlin, pp. 421–22. [15] Stryer et al., p. 749. [20] Stryer et al., pp. 329–331 [21] Heinz E.(1996). Plant glycolipids: structure, isolation and analysis. in Advances in Lipid Methodology - 3, pp. 211–332 (ed. W.W. Christie, Oily Press, Dundee) [22] Stryer et al., pp. 333–34. [26] Stryer et al., p. 619. [36] http:/ / www. elmhurst. edu/ ~chm/ vchembook/ 556steroids. html [37] Stryer et al., p. 634. [41] Stryer et al., p. 643. [42] Stryer et al., pp. 733–39. [46] Stryer et al., pp. 625–26. [47] Bhagavan, p. 903. [48] Bhagavan, p. 388.

Bibliography • Bhagavan NV (2002). Medical Biochemistry (http://books.google.com/?id=vT9YttFTPi0C& printsec=frontcover). San Diego: Harcourt/Academic Press. ISBN0-12-095440-0. • Devlin TM (1997). Textbook of Biochemistry: With Clinical Correlations (4th ed.). Chichester: John Wiley & Sons. ISBN0-471-17053-4. • Stryer L, Berg JM, Tymoczko JL (2007). Biochemistry (6th ed.). San Francisco: W.H. Freeman. ISBN0-7167-8724-5. • Van Holde KE, Mathews CK (1996). Biochemistry (2nd ed.). Menlo Park, Calif: Benjamin/Cummings Pub. Co. ISBN0-8053-3931-0.

External links Introductory • • • • •

List of lipid-related web sites (http://www.cyberlipid.org/cyberlip/link0041.htm) Nature Lipidomics Gateway (http://www.lipidmaps.org/) - Round-up and summaries of recent lipid research Lipid Library (http://www.lipidlibrary.co.uk/) - General reference on lipid chemistry and biochemistry Cyberlipid.org (http://www.cyberlipid.org/) - Resources and history for lipids. Molecular Computer Simulations (http://www.fos.su.se/~sasha/Lipid_membranes.html) - Modeling of Lipid Membranes • Lipids, Membranes and Vesicle Trafficking (http://www.biochemweb.org/lipids_membranes.shtml) - The Virtual Library of Biochemistry and Cell Biology Nomenclature • IUPAC nomenclature of lipids (http://www.chem.qmul.ac.uk/iupac/lipid/) • IUPAC glossary entry for the lipid class of molecules (http://www.chem.qmul.ac.uk/iupac/class/lipid.html) Databases • LIPID MAPS (http://www.lipidmaps.org/data/databases.html) - Comprehensive lipid and lipid-associated gene/protein databases.

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• LipidBank (http://lipidbank.jp/) - Japanese database of lipids and related properties, spectral data and references. • LIPIDAT (http://www.lipidat.tcd.ie/) - Database composed mainly of phospholipids and associated thermodynamic data. General • ApolloLipids (http://www.apollolipids.org/) - Provides dyslipidemia and cardiovascular disease prevention and treatment information as well as continuing medical education programs • National Lipid Association (http://www.lipid.org/) - Professional medical education organization for health care professionals who seek to prevent morbidity and mortality stemming from dyslipidemias and other cholesterol-related disorders.

Biological membrane A biological membrane or biomembrane is an enclosing or separating membrane that acts as a selective barrier, within or around a cell. It consists of a lipid bilayer with embedded proteins that may constitute close to 50% of membrane content.[1] The cellular membranes should not be confused with isolating tissues formed by layers of cells, such as mucous and basement membranes.

Function Membranes in cells typically define enclosed spaces or compartments in which cells may maintain a chemical or biochemical environment that differs from the outside. For example, the membrane around peroxisomes shields the rest of the cell from peroxides, and the cell membrane separates a cell from its surrounding medium. Most organelles are defined by such membranes, and are called "membrane-bound" organelles.

Cross section view of the structures that can be formed by phospholipids in aqueous solutions

Probably the most important feature of a biomembrane is that it is a selectively permeable structure. This means that the size, charge, and other chemical properties of the atoms and molecules attempting to cross it will determine whether they succeed in doing so. Selective permeability is essential for effective separation of a cell or organelle from its surroundings. Biological membranes also have certain mechanical or elastic properties. Particles that are required for cellular function but are unable to diffuse freely across a membrane enter through a membrane transport protein or are taken in by means of endocytosis.

Diversity of biological membranes Many types of specialized plasma membranes can separate cell from external environment: apical, basolateral, presynaptic and postsynaptic ones, membranes of flagella, cilia, microvillus, filopodia and lamellipodia, the sarcolemma of muscle cells, as well as specialized myelin and dendritic spine membranes of neurons. Plasma membranes can also form different types of "supramembrane" structures such as caveola, postsynaptic density, podosome, invadopodium, desmosome, hemidesmosome, focal adhesion, and cell junctions. These types of membranes differ in lipid and protein composition.

Biological membrane Distinct types of membranes also create intracellular organelles: endosome; smooth and rough endoplasmic reticulum; sarcoplasmic reticulum; Golgi apparatus; lysosome; mitochondrion (inner and outer membranes); nucleus (inner and outer membranes); peroxisome; vacuole; cytoplasmic granules; cell vesicles (phagosome, autophagosome, clathrin-coated vesicles, COPI-coated and COPII-coated vesicles) and secretory vesicles (including synaptosome, acrosomes, melanosomes, and chromaffin granules). Different types of biological membranes have diverse lipid and protein compositions. The content of membranes defines their physical and biological properties. Some components of membranes play a key role in medicine, such as the efflux pumps that pump drugs out of a cell.

References • von Heijne G, Rees D (August 2008). "Membranes: reading between the lines" (http://linkinghub.elsevier.com/ retrieve/pii/S0959-440X(08)00091-2). Curr. Opin. Struct. Biol. 18 (4): 403–5. doi: 10.1016/j.sbi.2008.06.003 (http://dx.doi.org/10.1016/j.sbi.2008.06.003). PMID 18634876 (http://www.ncbi.nlm.nih.gov/ pubmed/18634876).

External links • Membranes (http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=&term=Membranes) at the US National Library of Medicine Medical Subject Headings (MeSH)

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Membrane protein Membrane proteins constitute one of the three main protein classes, with the other classes being the fibrous and globular proteins. Membrane proteins are attached to, or associated with the membrane of a cell or an organelle. These proteins are specifically targeted to different types of biological membranes [1] They are also the target of over 50% of all modern medicinal drugs.[] It is estimated that 20-30% of all genes in most genomes encode membrane proteins.[2]

Function Membrane proteins perform a variety of functions vital to the survival of organisms:[3] • Membrane receptor proteins relay signals between the cell's internal and external environments. • Transport proteins move molecules and ions across the membrane. They can be categorised according to the Transporter Classification database. • Membrane enzymes for example Oxidoreductases, Transferases and Hydrolases.

Crystal structure of Potassium channel KvAP. Calculated hydrocarbon boundaries of the lipid bilayer are indicated by red and blue dots.

• Cell adhesion molecules allow cells to identify each other and interact. For example proteins involved in immune response.

Topology The topology of an integral membrane protein describes the number of transmembrane segments, as well as the orientation in the membrane.[4] Membrane proteins have several different topologies:[] A slightly different classification is to divide all membrane proteins to integral and amphitropic.[] The amphitropic are proteins that can exist in two alternative states: a water-soluble and a lipid bilayer-bound. The amphitropic protein category includes water-soluble channel-forming polypeptide toxins, which associate irreversibly with membranes, but excludes peripheral proteins that interact with other membrane proteins rather than with lipid bilayer.

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Integral membrane proteins Integral membrane proteins are permanently attached to the membrane. Such proteins can be separated from the biological membranes only using detergents, nonpolar solvents, or sometimes denaturing agents. They can be classified according to their relationship with the bilayer: • Integral polytopic proteins, also known as "transmembrane proteins," are integral membrane proteins which span across the membrane at least once. They have one of two tertiary structures: • Helix bundle proteins which are present in all types of biological membranes;

Schematic representation of transmembrane proteins: 1. a single transmembrane α-helix (bitopic membrane protein) 2. a polytopic transmembrane α-helical protein 3. a polytopic transmembrane β-sheet protein The membrane is represented in light brown.

• Beta barrel proteins which are found only in outer membranes of Gram-negative bacteria, lipid-rich cell walls of a few Gram-positive bacteria, and outer membranes of mitochondria and chloroplasts. • Integral monotopic proteins are integral membrane proteins which are attached to only one side of the membrane and do not span the whole way across.

Peripheral membrane proteins Peripheral membrane proteins are temporarily attached either to the lipid bilayer or to integral proteins by a combination of hydrophobic, electrostatic, and other non-covalent interactions. Peripheral proteins dissociate following treatment with a polar reagent, such as a solution with an elevated pH or high salt concentrations.

Schematic representation of the different types of interaction between monotopic membrane proteins and the cell membrane: 1. interaction by an amphipathic α-helix parallel to the membrane plane (in-plane membrane helix) 2. interaction by a hydrophobic loop 3. interaction by a covalently bound membrane lipid (lipidation) 4. electrostatic or ionic interactions with membrane lipids (e.g. through a calcium ion)

Integral and peripheral proteins may be post-translationally modified, with added fatty acid or prenyl chains, or GPI (glycosylphosphatidylinositol), which may be anchored in the lipid bilayer.

Polypeptide toxins Polypeptide toxins and many antibacterial peptides, such as colicins or hemolysins, and certain proteins involved in apoptosis, are sometimes considered a separate category. These proteins are water-soluble but can aggregate and associate irreversibly with the lipid bilayer and become reversibly or irreversibly membrane-associated.

Membrane protein

3D Structure The most common tertiary structures are Helix bundle and Beta barrel. The portion of the membrane proteins that are attached to the lipid bilayer are consisting of hydrophobic amino acids only. This is done so that the peptide bonds' carbonyl and amine will react with each other instead of the hydrophobic surrounding. The portion of the protein that is not touching the lipid bilayer and is protruding out of the cell membrane are usually hydrophilic amino acids.[5] Membrane proteins have hydrophobic surfaces, are relatively flexible and are expressed at relatively low levels. This creates difficulties in Increase in the number of 3D structures of obtaining enough protein and then growing crystals. Hence despite the membrane proteins known significant functional importance of membrane proteins, determining atomic resolution structures for these proteins is more difficult than globular proteins.[6] As of January 2013 less than 0.1% of protein structures determined were membrane proteins despite being 20-30% of the total proteome.[7] Many of the successful membrane protein structures are characterized by X-ray crystallography and are very large structures in which the interactions with the membrane mimetic environments can be anticipated to be small in comparison to those within the protein structures. The small domains are particularly sensitive to the influence of membrane mimetic environments, potentially leading to non-native structures. Fortunately, there are many sample preparation conditions that can be chosen for crystallization and for solution NMR. All membrane protein structural biology should be subjected to careful scrutiny; through a combination of structural methodologies it should be possible to achieve an understanding of the native functional state for membrane protein structures.[8] Coevolution information has been successfully exploited for prediction of multiple large (membrane) protein structures.[9][10][11] Due to this difficulty and the importance of this class of proteins methods of protein structure prediction based on hydropathy plots and the positive inside rule have been developed.[12][13]

References White, Stephen. “General Principle of Membrane Protein Folding and Stability.” Stephen White Laboratory Homepage. 10 Nov. 2009. web. [1] [5] [7] [8]

Classification of membrane proteins with known 3D structure to different membrane types (http:/ / opm. phar. umich. edu/ atlas. php) White, Stephen. “General Principle of Membrane Protein Folding and Stability.” Stephen White Laboratory Homepage. 10 Nov. 2009. web. Membrane Proteins of known 3D Structure (http:/ / blanco. biomol. uci. edu/ mpstruc/ ) Cross, Timothy, Mukesh Sharma, Myunggi Yi, Huan-Xiang Zhou (2010). "Influence of Solubilizing Environments on Membrane Protein Structures" [13] State of the art in membrane protein prediction (http:/ / gepard. bioinformatik. uni-saarland. de/ old_html/ html/ MembraneBioinformaticsSS06/ SuggestedReadingLecture6/ Chen_Review. pdf)

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External links Organizations • Membrane Protein Structural Dynamics Consortium (http://memprotein.org) • Transmembrane Protein Center (http://www.uwmembraneproteins.org)

Membrane protein databases • List of transmembrane proteins of known 3D structure (http://blanco.biomol.uci.edu/ Membrane_Proteins_xtal.html) • TCDB (http://www.tcdb.org/) - Transporter Classification database • Orientations of Proteins in Membranes (OPM) database (http://opm.phar.umich.edu/) 3D structures of integral and peripheral membrane proteins arranged in the lipid bilayer • Membrane PDB (http://www.mpdb.tcd.ie/) Database of 3D structures of integral membrane proteins and hydrophobic peptides with an emphasis on crystallization conditions • Protein Data Bank of Transmembrane Proteins (http://pdbtm.enzim.hu/) 3D models of all transmembrane proteins currently in PDB. Approximate positions of membrane boundary planes were calculated for each PDB entry. • TransportDB (http://www.membranetransport.org/) Genomics-oriented database of transporters from TIGR • Membrane targeting domains (MeTaDoR) (http://proteomics.bioengr.uic.edu/metador/MeTaDoR.html)

Further reading • The Human Membrane Proteome (http://www.biomedcentral.com/1741-7007/7/50) - A comprehensive article covering the transmembrane protein component of the human proteome

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Cell membrane The cell membrane is a biological membrane that separates the interior of all cells from the outside environment.[1] The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells.[] The basic function of the cell membrane is to protect the cell from its surroundings. It consists of the lipid bilayer with embedded proteins. Cell membranes are involved in a variety of cellular processes such as cell adhesion, ion conductivity and cell signaling and serve as the attachment surface for several extracellular structures, including the cell wall, glycocalyx, and intracellular cytoskeleton. Cell membranes can be artificially reassembled.[][][]

Illustration of a Eukaryotic cell membrane

Function The cell membrane or plasma membrane surrounds the cytoplasm of living cells, physically separating the intracellular components from the extracellular environment. Fungi, bacteria and plants also have the cell wall which provides a mechanical support for the cell and precludes the passage of larger molecules. The cell A detailed diagram of the cell membrane membrane also plays a role in anchoring the cytoskeleton to provide shape to the cell, and in attaching to the extracellular matrix and other cells to help group cells together to form tissues. The membrane is selectively permeable and able to regulate what enters and exits the cell, thus facilitating the transport of materials needed for survival. The movement of substances across the membrane can be either "passive", occurring without the input of cellular energy, or active, requiring the cell to expend energy in transporting it. The

Cell membrane membrane also maintains the cell potential. The cell membrane thus works as a selective filter that allows only certain things to come inside or go outside the cell. The cell employs a number of transport mechanisms that involve biological membranes: 1. Passive diffusion and osmosis: Some substances (small molecules, ions) such as carbon dioxide (CO2) and oxygen (O2), can move across the plasma membrane by diffusion, which is a passive transport process. Because the membrane acts as a barrier for certain molecules and ions, they can occur in different concentrations on the two sides of the membrane. Such a concentration gradient across a semipermeable membrane sets up an osmotic flow for the water. 2. Transmembrane protein channels and transporters: Nutrients, such as sugars or amino acids, must enter the cell, and certain products of metabolism must leave the cell. Such molecules are pumped across the membrane by transmembrane transporters or diffuse through protein channels such as Aquaporins (in the case of water (H2O)). These proteins, also called permeases, are usually quite specific, recognizing and transporting only a limited food group of chemical substances, often even only a single substance. 3. Endocytosis: Endocytosis is the process in which cells absorb molecules by engulfing them. The plasma membrane creates a small deformation inward, called an invagination, in which the substance to be transported is captured. The deformation then pinches off from the membrane on the inside of the cell, creating a vesicle containing the captured substance. Endocytosis is a pathway for internalizing solid particles (cell eating or phagocytosis), small molecules and ions (cell drinking or pinocytosis), and macromolecules. Endocytosis requires energy and is thus a form of active transport. 4. Exocytosis: Just as material can be brought into the cell by invagination and formation of a vesicle, the membrane of a vesicle can be fused with the plasma membrane, extruding its contents to the surrounding medium. This is the process of exocytosis. Exocytosis occurs in various cells to remove undigested residues of substances brought in by endocytosis, to secrete substances such as hormones and enzymes, and to transport a substance completely across a cellular barrier. In the process of exocytosis, the undigested waste-containing food vacuole or the secretory vesicle budded from Golgi apparatus, is first moved by cytoskeleton from the interior of the cell to the surface. The vesicle membrane comes in contact with the plasma membrane. The lipid molecules of the two bilayers rearrange themselves and the two membranes are, thus, fused. A passage is formed in the fused membrane and the vesicles discharges its contents outside the cell.

Prokaryotes Gram-negative bacteria have a plasma membrane and an outer membrane separated by a periplasmic space. Other prokaryotes have only a plasma membrane. Prokaryotic cells are also surrounded by a cell wall composed of peptidoglycan (amino acids and sugars). Some eukaryotic cells also have cells walls, but none that are made of peptidoglycan.

Structure Fluid mosaic model According to the fluid mosaic model of S.J. Singer and G.L. Nicolson (1972), which replaced the earlier model of Davson and Danielli, biological membranes can be considered as a two-dimensional liquid in which lipid and protein molecules diffuse more or less easily.[2] Although the lipid bilayers that form the basis of the membranes do indeed form two-dimensional liquids by themselves, the plasma membrane also contains a large quantity of proteins, which provide more structure. Examples of such structures are protein-protein complexes, pickets and fences formed by the actin-based cytoskeleton, and potentially lipid rafts.

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Lipid bilayer Lipid bilayers form through the process of self-assembly. The cell membrane consists primarily of a thin layer of amphipathic phospholipids which spontaneously arrange so that the hydrophobic "tail" regions are isolated from the surrounding polar fluid, causing the more hydrophilic "head" regions to associate with the intracellular (cytosolic) and extracellular faces of the resulting bilayer. This forms a continuous, spherical lipid bilayer. Forces such as van der Waals, electrostatic, hydrogen bonds, and noncovalent interactions all contribute to the formation of the lipid bilayer. Overall, hydrophobic interactions are the major driving force in the formation of lipid bilayers.

Diagram of the arrangement of amphipathic lipid molecules to form a lipid bilayer. The yellow polar head groups separate the grey hydrophobic tails from the aqueous cytosolic and extracellular environments.

Lipid bilayers are generally impermeable to ions and polar molecules. The arrangement of hydrophilic heads and hydrophobic tails of the lipid bilayer prevent polar solutes (ex. amino acids, nucleic acids, carbohydrates, proteins, and ions) from diffusing across the membrane, but generally allows for the passive diffusion of hydrophobic molecules. This affords the cell the ability to control the movement of these substances via transmembrane protein complexes such as pores, channels and gates. Flippases and scramblases concentrate phosphatidyl serine, which carries a negative charge, on the inner membrane. Along with NANA, this creates an extra barrier to charged moieties moving through the membrane. Membranes serve diverse functions in eukaryotic and prokaryotic cells. One important role is to regulate the movement of materials into and out of cells. The phospholipid bilayer structure (fluid mosaic model) with specific membrane proteins accounts for the selective permeability of the membrane and passive and active transport mechanisms. In addition, membranes in prokaryotes and in the mitochondria and chloroplasts of eukaryotes facilitate the synthesis of ATP through chemiosmosis.

Membrane polarity The apical membrane of a polarized cell is the surface of the plasma membrane that faces inward to the lumen. This is particularly evident in epithelial and endothelial cells, but also describes other polarized cells, such as neurons. The basolateral membrane of a polarized cell is the surface of the plasma membrane that forms its basal and lateral surfaces. It faces outwards, towards the interstitium, and away from the lumen. Basolateral membrane is a compound phrase referring to the terms "basal (base) membrane" and "lateral (side) membrane", which, especially Alpha intercalated cell in epithelial cells, are identical in composition and activity. Proteins (such as ion channels and pumps) are free to move from the basal to the lateral surface of the cell or vice versa in accordance with the fluid mosaic model. Tight junctions join epithelial cells near their apical surface to prevent the migration of proteins from the basolateral membrane to the apical membrane. The basal and lateral surfaces thus remain roughly

Cell membrane equivalentWikipedia:Please clarify to one another, yet distinct from the apical surface.

Membrane structures Cell membrane can form different types of "supramembrane" structures such as caveola, postsynaptic density, podosome, invadopodium, focal adhesion, and different types of cell junctions. These structures are usually responsible for cell adhesion, communication, endocytosis and exocytosis. They can be visualized by electron microscopy or fluorescence microscopy. They are composed of specific proteins, such as integrins and cadherins.

Cytoskeleton The cytoskeleton is found underlying the cell membrane in the cytoplasm and provides a scaffolding for membrane proteins to anchor to, as well as forming organelles that extend from the cell. Indeed, cytoskeletal elements interact extensively and intimately with the cell membrane.[3] Anchoring proteins restricts them to a particular cell surface — for example, the apical surface of epithelial cells that line the vertebrate gut — and limits how far they may diffuse within the bilayer. The cytoskeleton is able to form appendage-like organelles, such as cilia, which are microtubule-based extensions covered by the cell membrane, and filopodia, which are actin-based extensions. These extensions are ensheathed in membrane and project from the surface of the cell in order to sense the external environment and/or make contact with the substrate or other cells. The apical surfaces of epithelial cells are dense with actin-based finger-like projections known as microvilli, which increase cell surface area and thereby increase the absorption rate of nutrients. Localized decoupling of the cytoskeleton and cell membrane results in formation of a bleb.

Composition Cell membranes contain a variety of biological molecules, notably lipids and proteins. Material is incorporated into the membrane, or deleted from it, by a variety of mechanisms: • Fusion of intracellular vesicles with the membrane (exocytosis) not only excretes the contents of the vesicle but also incorporates the vesicle membrane's components into the cell membrane. The membrane may form blebs around extracellular material that pinch off to become vesicles (endocytosis). • If a membrane is continuous with a tubular structure made of membrane material, then material from the tube can be drawn into the membrane continuously. • Although the concentration of membrane components in the aqueous phase is low (stable membrane components have low solubility in water), there is an exchange of molecules between the lipid and aqueous phases.

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Lipids The cell membrane consists of three classes of amphipathic lipids: phospholipids, glycolipids, and cholesterols. The amount of each depends upon the type of cell, but in the majority of cases phospholipids are the most abundant.[] In RBC studies, 30% of the plasma membrane is lipid. The fatty chains in phospholipids and glycolipids usually contain an even number of carbon atoms, typically between 16 and 20. The 16- and 18-carbon fatty acids are the most common. Fatty acids may be saturated or unsaturated, with the configuration of the double bonds nearly always "cis". The length and the degree of unsaturation of fatty acid chains have a profound effect on membrane fluidity[] as unsaturated lipids create a kink, preventing the fatty Examples of the major membrane phospholipids and glycolipids: phosphatidylcholine acids from packing together as tightly, (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns), thus decreasing the melting temperature phosphatidylserine (PtdSer). (increasing the fluidity) of the membrane. The ability of some organisms to regulate the fluidity of their cell membranes by altering lipid composition is called homeoviscous adaptation. The entire membrane is held together via non-covalent interaction of hydrophobic tails, however the structure is quite fluid and not fixed rigidly in place. Under physiological conditions phospholipid molecules in the cell membrane are in the liquid crystalline state. It means the lipid molecules are free to diffuse and exhibit rapid lateral diffusion along the layer in which they are present. However, the exchange of phospholipid molecules between intracellular and extracellular leaflets of the bilayer is a very slow process. Lipid rafts and caveolae are examples of cholesterol-enriched microdomains in the cell membrane. In animal cells cholesterol is normally found dispersed in varying degrees throughout cell membranes, in the irregular spaces between the hydrophobic tails of the membrane lipids, where it confers a stiffening and strengthening effect on the membrane.[]

Phospholipids forming lipid vesicles Lipid vesicles or liposomes are circular pockets that are enclosed by a lipid bilayer. These structures are used in laboratories to study the effects of chemicals in cells by delivering these chemicals directly to the cell, as well as getting more insight into cell membrane permeability. Lipid vesicles and liposomes are formed by first suspending a lipid in an aqueous solution then agitating the mixture through sonication, resulting in a vesicle. By measuring the rate of efflux from that of the inside of the vesicle to the ambient solution, allows researcher to better understand membrane permeability. Vesicles can be formed with molecules and ions inside the vesicle by forming the vesicle with the desired molecule or ion present in the solution. Proteins can also be embedded into the membrane through solubilizing the desired proteins in the presence of detergents and attaching them to the phospholipids in which the

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liposome is formed. These provide researchers with a tool to examine various membrane protein functions.

Carbohydrates Plasma membranes also contain carbohydrates, predominantly glycoproteins, but with some glycolipids (cerebrosides and gangliosides). For the most part, no glycosylation occurs on membranes within the cell; rather generally glycosylation occurs on the extracellular surface of the plasma membrane. The glycocalyx is an important feature in all cells, especially epithelia with microvilli. Recent data suggest the glycocalyx participates in cell adhesion, lymphocyte homing, and many others. The penultimate sugar is galactose and the terminal sugar is sialic acid, as the sugar backbone is modified in the golgi apparatus. Sialic acid carries a negative charge, providing an external barrier to charged particles.

Proteins Type

Description

Examples

Integral proteins or transmembrane proteins

Span the membrane and have a hydrophilic cytosolic domain, which interacts with internal molecules, a hydrophobic membrane-spanning domain that anchors it within the cell membrane, and a hydrophilic extracellular domain that interacts with external molecules. The hydrophobic domain consists of one, multiple, or a combination of α-helices and β sheet protein motifs.

Ion channels, proton pumps, G protein-coupled receptor

Lipid anchored proteins

Covalently bound to single or multiple lipid molecules; hydrophobically insert into the cell membrane and anchor the protein. The protein itself is not in contact with the membrane.

G proteins

Peripheral proteins

Attached to integral membrane proteins, or associated with peripheral regions of the lipid bilayer. These proteins tend to have only temporary interactions with biological membranes, and once reacted, the molecule dissociates to carry on its work in the cytoplasm.

Some enzymes, some hormones

The cell membrane has large content of proteins, typically around 50% of membrane volume[] These proteins are important for cell because they are responsible for various biological activities. Approximately a third of the genes in yeast code specifically for them, and this number is even higher in multicellular organisms.[] The cell membrane, being exposed to the outside environment, is an important site of cell–cell communication. As such, a large variety of protein receptors and identification proteins, such as antigens, are present on the surface of the membrane. Functions of membrane proteins can also include cell–cell contact, surface recognition, cytoskeleton contact, signaling, enzymatic activity, or transporting substances across the membrane. Most membrane proteins must be inserted in some way into the membrane. For this to occur, an N-terminus "signal sequence" of amino acids directs proteins to the endoplasmic reticulum, which inserts the proteins into a lipid bilayer. Once inserted, the proteins are then transported to their final destination in vesicles, where the vesicle fuses with the target membrane.

Variation The cell membrane has different lipid and protein compositions in distinct types of cells and may have therefore specific names for certain cell types: • • • •

Sarcolemma in myocytes Oolemma in oocytes Axolemma in neuronal processes - axons Historically, the plasma membrane was also referred to as the plasmalemma

Cell membrane

Permeability The permeability of a membrane is the rate of passive diffusion of molecules through the membrane. These molecules are known as permeant molecules. Permeability depends mainly on the electric charge and polarity of the molecule and to a lesser extent the molar mass of the molecule. Due to the cell membrane's hydrophobic nature, small electrically neutral molecules pass through the membrane more easily than charged, large ones. The inability of charged molecules to pass through the cell membrane results in pH partition of substances throughout the fluid compartments of the body.

References [1] Kimball's Biology Pages (http:/ / users. rcn. com/ jkimball. ma. ultranet/ BiologyPages/ C/ CellMembranes. html), Cell Membranes

External links • Lipids, Membranes and Vesicle Trafficking - The Virtual Library of Biochemistry and Cell Biology (http:// www.biochemweb.org/lipids_membranes.shtml) • Cell membrane protein extraction protocol (http://www.westernblotting.org/protocol membrane extraction. htm) • Membrane homeostasis, tension regulation, mechanosensitive membrane exchange and membrane traffic (http:// www.phys.unsw.edu.au/~jw/tension.html) • 3D structures of proteins associated with plasma membrane of eukaryotic cells (http://opm.phar.umich.edu/ localization.php?localization=Eukaryotic plasma membrane) • Lipid composition and proteins of some eukariotic membranes (http://opm.phar.umich.edu/atlas. php?membrane=Eukaryotic plasma membrane) • (http://www.etap.org/demo/biology1/instruction3tutor.html)

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Carbohydrate structure Carbohydrate A carbohydrate is an organic compound that consists only of carbon, hydrogen, and oxygen, usually with a hydrogen:oxygen atom ratio of 2:1 (as in water); in other words, with the empirical formula Cm(H2O)n (where m could be different from n). Some exceptions exist; for example, deoxyribose, a component of DNA, has the empirical formula C5H10O4. Carbohydrates are not technically hydrates of carbon; structurally it is more accurate to view them as polyhydroxy aldehydes and ketones.

Lactose is a disaccharide found in milk. It consists of a molecule of D-galactose and a molecule of D-glucose bonded by beta-1-4 glycosidic linkage. It has a formula of C12H22O11.

The term is most common in biochemistry, where it is a synonym of saccharide. The carbohydrates (saccharides) are divided into four chemical groupings: monosaccharides, disaccharides, oligosaccharides, and polysaccharides. In general, the monosaccharides and disaccharides, which are smaller (lower molecular weight) carbohydrates, are commonly referred to as sugars.[1] The word saccharide comes from the Greek word σάκχαρον (sákkharon), meaning "sugar." While the scientific nomenclature of carbohydrates is complex, the names of the monosaccharides and disaccharides very often end in the suffix -ose. For example, blood sugar is the monosaccharide glucose, table sugar is the disaccharide sucrose, and milk sugar is the disaccharide lactose (see illustration). Carbohydrates perform numerous roles in living organisms. Polysaccharides serve for the storage of energy (e.g., starch and glycogen), and as structural components (e.g., cellulose in plants and chitin in arthropods). The 5-carbon monosaccharide ribose is an important component of coenzymes (e.g., ATP, FAD, and NAD) and the backbone of the genetic molecule known as RNA. The related deoxyribose is a component of DNA. Saccharides and their derivatives include many other important biomolecules that play key roles in the immune system, fertilization, preventing pathogenesis, blood clotting, and development.[2] In food science and in many informal contexts, the term carbohydrate often means any food that is particularly rich in the complex carbohydrate starch (such as cereals, bread, and pasta) or simple carbohydrates, such as sugar (found in candy, jams, and desserts).

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Structure Formerly the name "carbohydrate" was used in chemistry for any compound with the formula Cm (H2O) n. Following this definition, some chemists considered formaldehyde (CH2O) to be the simplest carbohydrate,[3] while others claimed that title for glycolaldehyde.[4] Today the term is generally understood in the biochemistry sense, which excludes compounds with only one or two carbons. Natural saccharides are generally built of simple carbohydrates called monosaccharides with general formula (CH2O)n where n is three or more. A typical monosaccharide has the structure H-(CHOH)x(C=O)-(CHOH)y-H, that is, an aldehyde or ketone with many hydroxyl groups added, usually one on each carbon atom that is not part of the aldehyde or ketone functional group. Examples of monosaccharides are glucose, fructose, and glyceraldehydes. However, some biological substances commonly called "monosaccharides" do not conform to this formula (e.g., uronic acids and deoxy-sugars such as fucose), and there are many chemicals that do conform to this formula but are not considered to be monosaccharides (e.g., formaldehyde CH2O and inositol (CH2O)6).[5] The open-chain form of a monosaccharide often coexists with a closed ring form where the aldehyde/ketone carbonyl group carbon (C=O) and hydroxyl group (-OH) react forming a hemiacetal with a new C-O-C bridge. Monosaccharides can be linked together into what are called polysaccharides (or oligosaccharides) in a large variety of ways. Many carbohydrates contain one or more modified monosaccharide units that have had one or more groups replaced or removed. For example, deoxyribose, a component of DNA, is a modified version of ribose; chitin is composed of repeating units of N-acetyl glucosamine, a nitrogen-containing form of glucose.

Monosaccharides Monosaccharides are the simplest carbohydrates in that they cannot be hydrolyzed to smaller carbohydrates. They are aldehydes or ketones with two or more hydroxyl groups. The general chemical formula of an unmodified monosaccharide is (C•H2O) n, literally a "carbon hydrate." Monosaccharides are important fuel molecules as well as building blocks for nucleic acids. The smallest monosaccharides, for which n=3, are dihydroxyacetone and D- and L-glyceraldehydes.

Classification of monosaccharides

The α and β anomers of glucose. Note the position of the hydroxyl group (red or green) on the anomeric carbon relative to the CH2OH group bound to carbon 5: they are either on the opposite sides (α), or the same side (β).

D-glucose is an aldohexose with the formula (C·H2O)6. The red atoms highlight the aldehyde group, and the blue atoms highlight the asymmetric center furthest from the aldehyde; because this -OH is on the right of the Fischer projection, this is a D sugar.

Monosaccharides are classified according to three different characteristics: the placement of its carbonyl group, the number of carbon atoms it contains, and its chiral handedness. If the carbonyl group is an aldehyde, the monosaccharide is an aldose; if the carbonyl group is a ketone, the monosaccharide is a ketose. Monosaccharides with three carbon atoms are called trioses, those with four are called tetroses, five are called pentoses, six are hexoses, and so on.[6] These two systems of classification are often combined. For example, glucose is an aldohexose (a six-carbon aldehyde), ribose is an aldopentose (a five-carbon aldehyde), and fructose is a ketohexose (a six-carbon ketone).

Carbohydrate Each carbon atom bearing a hydroxyl group (-OH), with the exception of the first and last carbons, are asymmetric, making them stereo centers with two possible configurations each (R or S). Because of this asymmetry, a number of isomers may exist for any given monosaccharide formula. The aldohexose D-glucose, for example, has the formula (C·H2O) 6, of which all but two of its six carbons atoms are stereogenic, making D-glucose one of 24=16 possible stereoisomers. In the case of glyceraldehydes, an aldotriose, there is one pair of possible stereoisomers, which are enantiomers and epimers. 1, 3-dihydroxyacetone, the ketose corresponding to the aldose glyceraldehydes, is a symmetric molecule with no stereo centers). The assignment of D or L is made according to the orientation of the asymmetric carbon furthest from the carbonyl group: in a standard Fischer projection if the hydroxyl group is on the right the molecule is a D sugar, otherwise it is an L sugar. The "D-" and "L-" prefixes should not be confused with "d-" or "l-", which indicate the direction that the sugar rotates plane polarized light. This usage of "d-" and "l-" is no longer followed in carbohydrate chemistry.[7]

Ring-straight chain isomerism The aldehyde or ketone group of a straight-chain monosaccharide will react reversibly with a hydroxyl group on a different carbon atom to form a hemiacetal or hemiketal, forming a heterocyclic ring with an oxygen bridge between two carbon atoms. Rings with five and six atoms are called furanose and pyranose forms, respectively, and exist in equilibrium with the straight-chain form.[] During the conversion from straight-chain form to the cyclic form, the carbon atom containing the carbonyl oxygen, called the anomeric carbon, becomes a stereogenic center with two possible Glucose can exist in both a straight-chain and ring configurations: The oxygen atom may take a position either above form. or below the plane of the ring. The resulting possible pair of stereoisomers is called anomers. In the α anomer, the -OH substituent on the anomeric carbon rests on the opposite side (trans) of the ring from the CH2OH side branch. The alternative form, in which the CH2OH substituent and the anomeric hydroxyl are on the same side (cis) of the plane of the ring, is called the β anomer.

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Use in living organisms Monosaccharides are the major source of fuel for metabolism, being used both as an energy source (glucose being the most important in nature) and in biosynthesis. When monosaccharides are not immediately needed by many cells they are often converted to more space-efficient forms, often polysaccharides. In many animals, including humans, this storage form is glycogen, especially in liver and muscle cells. In plants, starch, is used for the same purpose.

Disaccharides Two joined monosaccharides are called a disaccharide and these are the simplest polysaccharides. Examples include sucrose and lactose. They are composed of two monosaccharide units bound together by a covalent bond known as a glycosidic linkage formed via a dehydration reaction, resulting in the loss of a hydrogen atom from one monosaccharide and a hydroxyl group from the other. The formula of unmodified disaccharides is C12H22O11. Although there are numerous kinds of disaccharides, a handful of disaccharides are particularly notable.

Sucrose, also known as table sugar, is a common disaccharide. It is composed of two monosaccharides: D-glucose (left) and D-fructose (right).

Sucrose, pictured to the right, is the most abundant disaccharide, and the main form in which carbohydrates are transported in plants. It is composed of one D-glucose molecule and one D-fructose molecule. The systematic name for sucrose, O-α-D-glucopyranosyl-(1→2)-D-fructofuranoside, indicates four things: • Its monosaccharides: glucose and fructose • Their ring types: glucose is a pyranose, and fructose is a furanose • How they are linked together: the oxygen on carbon number 1 (C1) of α-D-glucose is linked to the C2 of D-fructose. • The -oside suffix indicates that the anomeric carbon of both monosaccharides participates in the glycosidic bond. Lactose, a disaccharide composed of one D-galactose molecule and one D-glucose molecule, occurs naturally in mammalian milk. The systematic name for lactose is O-β-D-galactopyranosyl-(1→4)-D-glucopyranose. Other notable disaccharides include maltose (two D-glucoses linked α-1,4) and cellulobiose (two D-glucoses linked β-1,4). Disaccharides can be classified into two types.They are reducing and non-reducing disaccharides. If the functional group is present in bonding with another sugar unit, it is called a reducing disaccharide or biose.

Carbohydrate

Nutrition Foods high in carbohydrate include fruits, sweets, soft drinks, breads, pastas, beans, potatoes, bran, rice, and cereals. Carbohydrates are a common source of energy in living organisms; however, no carbohydrate is an essential nutrient in humans.[] Carbohydrates are not necessary building blocks of other molecules, and the body can obtain all its energy from protein and fats.[][8] The brain and neurons generally cannot burn fat for energy, but use glucose or ketones. Humans can synthesize some glucose (in a set of processes known as gluconeogenesis) from specific amino acids, from the glycerol backbone in triglycerides and in some cases from fatty acids. Carbohydrate and protein contain 4 calories per gram, while fats contain 9 calories per gram. In the case of protein, this is somewhat misleading as only some amino acids are usable for fuel. Organisms typically cannot metabolize all types of carbohydrate to yield energy. Glucose is a nearly universal and accessible source of Grain products: rich sources of carbohydrates calories. Many organisms also have the ability to metabolize other monosaccharides and Disaccharides, though glucose is preferred. In Escherichia coli, for example, the lac operon will express enzymes for the digestion of lactose when it is present, but if both lactose and glucose are present the lac operon is repressed, resulting in the glucose being used first (see: Diauxie). Polysaccharides are also common sources of energy. Many organisms can easily break down starches into glucose, however, most organisms cannot metabolize cellulose or other polysaccharides like chitin and arabinoxylans. These carbohydrates types can be metabolized by some bacteria and protists. Ruminants and termites, for example, use microorganisms to process cellulose. Even though these complex carbohydrates are not very digestible, they represent an important dietary element for humans, called dietary fiber. Fiber enhances digestion, among other benefits.[9] Based on the effects on risk of heart disease and obesity,[10] the Institute of Medicine recommends that American and Canadian adults get between 45–65% of dietary energy from carbohydrates.[11] The Food and Agriculture Organization and World Health Organization jointly recommend that national dietary guidelines set a goal of 55–75% of total energy from carbohydrates, but only 10% directly from sugars (their term for simple carbohydrates).[12]

Classification Nutritionists often refer to carbohydrates as either simple or complex. However, the exact delineation of these categories can be ambiguous. The term complex carbohydrate was first used in the U.S. Senate Select Committee on Nutrition and Human Needs publication Dietary Goals for the United States (1977) where it was intended to distinguish sugars from other carbohydrates (which were perceived to be nutritionally superior).[13] However, the report put "fruit, vegetables and whole-grains" in the complex carbohydrate column, despite the fact that these may contain sugars as well as polysaccharides. This confusion persists as today some nutritionists use the term complex carbohydrate to refer to any sort of digestible saccharide present in a whole food, where fiber, vitamins and minerals are also found (as opposed to processed carbohydrates, which provide calories but few other nutrients). The standard usage, however, is to classify carbohydrates chemically: simple if they are sugars (monosaccharides and disaccharides) and complex if they are polysaccharides (or oligosaccharides).[] In any case, the simple vs. complex chemical distinction has little value for determining the nutritional quality of carbohydrates.[] Some simple carbohydrates (e.g. fructose) are digested very slowly, while some complex

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Carbohydrate carbohydrates (starches), especially if processed, raise blood sugar rapidly. The speed of digestion is determined by a variety of factors including which other nutrients are consumed with the carbohydrate, how the food is prepared, individual differences in metabolism, and the chemistry of the carbohydrate.[] The USDA's Dietary Guidelines for Americans 2010 call for moderate- to high-carbohydrate consumption from a balanced diet that includes six one-ounce servings of grain foods each day, at least half from whole grain sources and the rest from enriched.[14] The glycemic index (GI) and glycemic load concepts have been developed to characterize food behavior during human digestion. They rank carbohydrate-rich foods based on the rapidity and magnitude of their effect on blood glucose levels. Glycemic index is a measure of how quickly food glucose is absorbed, while glycemic load is a measure of the total absorbable glucose in foods. The insulin index is a similar, more recent classification method that ranks foods based on their effects on blood insulin levels, which are caused by glucose (or starch) and some amino acids in food.

Metabolism Catabolism Catabolism is the metabolic reaction which cells undergo to extract energy. There are two major metabolic pathways of monosaccharide catabolism: glycolysis and the citric acid cycle. In glycolysis, oligo/polysaccharides are cleaved first to smaller monosaccharides by enzymes called glycoside hydrolases. The monosaccharide units can then enter into monosaccharide catabolism. In some cases, as with humans, not all carbohydrate types are usable as the digestive and metabolic enzymes necessary are not present.

Carbohydrate chemistry Carbohydrate chemistry is a large and economically important branch of organic chemistry. Some of the main organic reactions that involve carbohydrates are: • • • • • • •

Carbohydrate acetalisation Cyanohydrin reaction Lobry-de Bruyn-van Ekenstein transformation Amadori rearrangement Nef reaction Wohl degradation Koenigs–Knorr reaction

References [3] John Merle Coulter, Charler Reid Barnes, Henry Chandler Cowles (1930), A Textbook of Botany for Colleges and Universities (http:/ / books. google. com. br/ books?id=WyZnVpCiTHIC& pg=PA375& dq=simplest+ carbohydrate)" [4] Carl A. Burtis, Edward R. Ashwood, Norbert W. Tietz (2000), Tietz fundamentals of clinical chemistry (http:/ / books. google. com/ books?id=l5hqAAAAMAAJ& q=simplest+ carbohydrate) [5] Matthews, C. E.; K. E. Van Holde; K. G. Ahern (1999) Biochemistry. 3rd edition. Benjamin Cummings. ISBN 0-8053-3066-6 [11] Food and Nutrition Board (2002/2005). Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids (http:/ / newton. nap. edu/ books/ 0309085373/ html). Washington, D.C.: The National Academies Press. Page 769 (http:/ / newton. nap. edu/ books/ 0309085373/ html/ 769. html). ISBN 0-309-08537-3. [12] Joint WHO/FAO expert consultation (2003). (http:/ / www. webcitation. org/ query?id=1304266103156369) (PDF). Geneva: World Health Organization. pp. 55–56. ISBN 92-4-120916-X. [13] Joint WHO/FAO expert consultation (1998), Carbohydrates in human nutrition, chapter 1 (http:/ / www. fao. org/ docrep/ W8079E/ w8079e07. htm). ISBN 92-5-104114-8. [14] DHHS and USDA, Dietary Guidelines for Americans 2010 (http:/ / www. cnpp. usda. gov/ DietaryGuidelines. htm).

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External links • Carbohydrates, including interactive models and animations (http://www2.ufp.pt/~pedros/bq/carb_en.htm) (Requires MDL Chime (http://www.mdl.com/products/framework/chime/)) • IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (JCBN): Carbohydrate Nomenclature (http:// www.chem.qmw.ac.uk/iupac/2carb/) • Carbohydrates detailed (http://www.cem.msu.edu/~reusch/VirtualText/carbhyd.htm) • Complex And Simple Carbohydrates (http://evilcyber.com/nutrition/complex-and-simple-carbohydrates/) Explanation of the differences • Carbohydrates and Glycosylation – The Virtual Library of Biochemistry and Cell Biology (http://www. biochemweb.org/carbohydrates.shtml) • Functional Glycomics Gateway (http://www.functionalglycomics.org/), a collaboration between the Consortium for Functional Glycomics and Nature Publishing Group • Wine Carbohydrates (http://www.wineclubwizard.com/wine-carbohydrates.html)

Polysaccharide Polysaccharides are long carbohydrate molecules of monosaccharide units joined together by glycosidic bonds. They range in structure from linear to highly branched. Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating unit. Depending on the structure, these macromolecules can have distinct properties from their monosaccharide building blocks. They may be amorphous or even insoluble in water.[][]

3D structure of cellulose, a beta-glucan polysaccharide.

When all the monosaccharides in a polysaccharide are the same type, the polysaccharide is called a homopolysaccharide or homoglycan, but when more than one type of monosaccharide is present they are called heteropolysaccharides or heteroglycans.[1][2] Examples include storage polysaccharides such as starch and glycogen, and structural polysaccharides such as cellulose and chitin. Polysaccharides have a general formula of Cx(H2O)y where x is usually a large number between 200 and 2500. Considering that the repeating units in the polymer backbone are often six-carbon monosaccharides, the general formula can also be represented as (C6H10O5)n where 40≤n≤3000.

Polysaccharide

Structure Natural saccharides are generally built of simple carbohydrates called monosaccharides with general formula (CH2O)n where n is three or more. A typical monosaccharide has the structure H-(CHOH)x(C=O)-(CHOH)y-H, that is, an aldehyde or ketone with many hydroxyl groups added, usually one on each carbon atom that is not part of the aldehyde or ketone functional group. Examples of monosaccharides are glucose, fructose, and glyceraldehyde[3] Polysaccharides are composed of long chains of monosaccharide units bound together by glycosidic bonds. Polysaccharides contain more than ten monosaccharide units. Definitions of how large a carbohydrate must be to fall into the categories polysaccharides or oligosaccharides vary according to personal opinion. Polysaccharides is an important class Amylose is a linear polymer of glucose mainly linked with α(1→4) bonds. It can be made of biological polymers. Their function of several thousands of glucose units. It is one of the two components of starch, the other in living organisms is usually either being amylopectin. structure- or storage-related. Starch (a polymer of glucose) is used as a storage polysaccharide in plants, being found in the form of both amylose and the branched amylopectin. In animals, the structurally similar glucose polymer is the more densely branched glycogen, sometimes called 'animal starch'. Glycogen's properties allow it to be metabolized more quickly, which suits the active lives of moving animals. Cellulose and chitin are examples of structural polysaccharides. Cellulose is used in the cell walls of plants and other organisms, and is said to be the most abundant organic molecule on earth.[4] It has many uses such as a significant role in the paper and textile industries, and is used as a feedstock for the production of rayon (via the viscose process), cellulose acetate, celluloid, and nitrocellulose. Chitin has a similar structure, but has nitrogen-containing side branches, increasing its strength. It is found in arthropod exoskeletons and in the cell walls of some fungi. It also has multiple uses, including surgical threads. Polysaccharides also include callose or laminarin, chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan and galactomannan.

Function Nutrition Polysaccharides are common sources of energy. Many organisms can easily break down starches into glucose, however, most organisms cannot metabolize cellulose or other polysaccharides like chitin and arabinoxylans. These carbohydrates types can be metabolized by some bacteria and protists. Ruminants and termites, for example, use microorganisms to process cellulose. Even though these complex carbohydrates are not very digestible, they may comprise important dietary elements for humans. Called dietary fiber, these carbohydrates enhance digestion among other benefits. The main action of dietary fiber is to change the nature of the contents of the gastrointestinal tract, and to change how other nutrients and chemicals are absorbed.[][] Soluble fiber binds to bile acids in the small intestine, making them less likely to enter the body; this in turn lowers cholesterol levels in the blood.[] Soluble fiber also attenuates the absorption of sugar, reduces sugar response after eating, normalizes blood lipid levels and, once fermented in the colon, produces short-chain fatty acids as byproducts with wide-ranging physiological activities (discussion below). Although

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insoluble fiber is associated with reduced diabetes risk, the mechanism by which this occurs is unknown.[5] Not yet formally proposed as an essential macronutrient, dietary fiber is nevertheless regarded as important for the diet, with regulatory authorities in many developed countries recommending increases in fiber intake.[][][6][7]

Storage polysaccharides Starches Starches are glucose polymers in which glucopyranose units are bonded by alpha-linkages. It is made up of a mixture of amylose (15–20%) and amylopectin (80–85%). Amylose consists of a linear chain of several hundred glucose molecules and Amylopectin is a branched molecule made of several thousand glucose units (every chain of 24–30 glucose units is one unit of Amylopectin). Starches are insoluble in water. They can be digested by hydrolysis, catalyzed by enzymes called amylases, which can break the alpha-linkages (glycosidic bonds). Humans and other animals have amylases, so they can digest starches. Potato, rice, wheat, and maize are major sources of starch in the human diet. The formations of starches are the ways that plants store glucose.

Glycogen Glycogen serves as the secondary long-term energy storage in animal and fungal cells, with the primary energy stores being held in adipose tissue. Glycogen is made primarily by the liver and the muscles, but can also be made by glycogenesis within the brain and stomach.[9] Glycogen is the analogue of starch, a glucose polymer in plants, and is sometimes referred to as animal starch, having a similar structure to amylopectin but more extensively branched and compact than starch. Glycogen is a polymer of α(1→4) glycosidic bonds linked, with α(1→6)-linked branches. Glycogen is found in the form of granules in the cytosol/cytoplasm in many cell types, and plays an important role in the glucose cycle. Glycogen forms an energy reserve that can be quickly mobilized to meet a sudden need for glucose, but one that is less compact than the less immediately available energy reserves of triglycerides (lipids).

Schematic 2-D cross-sectional view of glycogen. A core protein of glycogenin is surrounded by branches of glucose units. The entire globular granule may contain [8] approximately 30,000 glucose units.

In the liver hepatocytes, glycogen can compose up to eight percent (100–120g in an adult) of the fresh weight soon after a meal.[] Only the glycogen stored in the liver

Polysaccharide

can be made accessible to other organs. In the muscles, glycogen is found in a low concentration of one to two percent of the muscle mass. The amount of glycogen stored in the body—especially within the muscles, liver, and red blood cells[10][11][12]—varies with physical activity, basal metabolic rate, and eating habits such as intermittent fasting. Small amounts of glycogen are found in the kidneys, and even smaller amounts in certain glial cells in the brain and white blood cells. The uterus also stores glycogen during pregnancy, to nourish the embryo.[] Glycogen is composed of a branched chain of glucose residues. It is stored in liver and muscles. • It is an energy reserve for animals. A view of the atomic structure of a single branched strand of glucose units in a • It is the chief form of carbohydrate stored glycogen molecule. in animal body. • It is insoluble in water. It turns red when mixed with iodine. • It also yields glucose on hydrolysis.

Structural polysaccharides Arabinoxylans Arabinoxylans are found in both the primary and secondary cell walls of plants and are the copolymers of two pentose sugars: arabinose and xylose.

Cellulose The structural component of plants are formed primarily from cellulose. Wood is largely cellulose and lignin, while paper and cotton are nearly pure cellulose. Cellulose is a polymer made with repeated glucose units bonded together by beta-linkages. Humans and many other animals lack an enzyme to break the beta-linkages, so they do not digest cellulose. Certain animals such as termites can digest cellulose, because bacteria possessing the enzyme are present in their gut. Cellulose is insoluble in water. It does not change color when mixed with iodine. On hydrolysis, it yields glucose. It is the most abundant carbohydrate in nature.

Chitin Chitin is one of many naturally occurring polymers. It forms a structural component of many animals, such as exoskeletons. Over time it is bio-degradable in the natural environment. Its breakdown may be catalyzed by enzymes called chitinases, secreted by microorganisms such as bacteria and fungi, and produced by some plants. Some of these microorganisms have receptors to simple sugars from the decomposition of chitin. If chitin is detected, they then produce enzymes to digest it by cleaving the glycosidic bonds in order to convert it to simple sugars and ammonia.

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Chemically, chitin is closely related to chitosan (a more water-soluble derivative of chitin). It is also closely related to cellulose in that it is a long unbranched chain of glucose derivatives. Both materials contribute structure and strength, protecting the organism.

Pectins Pectins are a family of complex polysaccharides that contain 1,4-linked α-D-galactosyluronic acid residues. They are present in most primary cell walls and in the non-woody parts of terrestrial plants.

Acidic polysaccharides Acidic polysaccharides are polysaccharides that contain carboxyl groups, phosphate groups and/or sulfuric ester groups.

Bacterial capsular polysaccharides Pathogenic bacteria commonly produce a thick, mucous-like, layer of polysaccharide. This "capsule" cloaks antigenic proteins on the bacterial surface that would otherwise provoke an immune response and thereby lead to the destruction of the bacteria. Capsular polysaccharides are water soluble, commonly acidic, and have molecular weights on the order of 100-2000 kDa. They are linear and consist of regularly repeating subunits of one to six monosaccharides. There is enormous structural diversity; nearly two hundred different polysaccharides are produced by E. coli alone. Mixtures of capsular polysaccharides, either conjugated or native are used as vaccines. Bacteria and many other microbes, including fungi and algae, often secrete polysaccharides as an evolutionary adaptation to help them adhere to surfaces and to prevent them from drying out. Humans have developed some of these polysaccharides into useful products, including xanthan gum, dextran, welan gum, gellan gum, diutan gum and pullulan. Most of these polysaccharides exhibit useful visco-elastic properties when dissolved in water at very low levels.[13] This makes various liquids used in everyday life, such as some foods, lotions, cleaners, and paints, viscous when stationary, but much more free-flowing when even slight shear is applied by stirring or shaking, pouring, wiping, or brushing. This property is named pseudoplasticity or shear thinning; the study of such matters is called rheology.

Viscosity of Welan gum [14] Shear Rate (rpm) Viscosity (cP) 0.3

23330

0.5

16000

11000

5500

3250

2900

1700

900

520

100

310

Aqueous solutions of the polysaccharide alone have a curious behavior when stirred: after stirring ceases, the solution initially continues to swirl due to momentum, then slows to a standstill due to viscosity and reverses

Polysaccharide direction briefly before stopping. This recoil is due to the elastic effect of the polysaccharide chains, previously stretched in solution, returning to their relaxed state. Cell-surface polysaccharides play diverse roles in bacterial ecology and physiology. They serve as a barrier between the cell wall and the environment, mediate host-pathogen interactions, and form structural components of biofilms. These polysaccharides are synthesized from nucleotide-activated precursors (called nucleotide sugars) and, in most cases, all the enzymes necessary for biosynthesis, assembly and transport of the completed polymer are encoded by genes organized in dedicated clusters within the genome of the organism. Lipopolysaccharide is one of the most important cell-surface polysaccharides, as it plays a key structural role in outer membrane integrity, as well as being an important mediator of host-pathogen interactions. The enzymes that make the A-band (homopolymeric) and B-band (heteropolymeric) O-antigens have been identified and the metabolic pathways defined.[15] The exopolysaccharide alginate is a linear copolymer of β-1,4-linked D-mannuronic acid and L-guluronic acid residues, and is responsible for the mucoid phenotype of late-stage cystic fibrosis disease. The pel and psl loci are two recently discovered gene clusters that also encode exopolysaccharides found to be important for biofilm formation. Rhamnolipid is a biosurfactant whose production is tightly regulated at the transcriptional level, but the precise role that it plays in disease is not well understood at present. Protein glycosylation, particularly of pilin and flagellin, became a focus of research by several groups from about 2007, and has been shown to be important for adhesion and invasion during bacterial infection.[]

Chemical identification tests for polysaccharides Periodic acid-Schiff stain (PAS) Polysaccharides with unprotected vicinal diols or amino sugars (i.e. some OH groups replaced with amine) give a positive Periodic acid-Schiff stain (PAS). The list of polysaccharides that stain with PAS is long. Although mucins of epithelial origins stain with PAS, mucins of connective tissue origin have so many acidic substitutions that they do not have enough glycol or amino-alcohol groups left to react with PAS.

References [3] Matthews, C. E.; K. E. Van Holde; K. G. Ahern (1999) Biochemistry. 3rd edition. Benjamin Cummings. ISBN 0-8053-3066-6 [4] N.A.Campbell (1996) Biology (4th edition). Benjamin Cummings NY. p.23 ISBN 0-8053-1957-3 [8] Page 12 in: (http:/ / books. google. dk/ books?id=SRptlOx7yj4C& printsec=frontcover& hl=en) Exercise physiology: energy, nutrition, and human performance By William D. McArdle, Frank I. Katch, Victor L. Katch Edition: 6, illustrated Published by Lippincott Williams & Wilkins, 2006 ISBN 0-7817-4990-5, ISBN 978-0-7817-4990-9, 1068 pages [9] Anatomy and Physiology. Saladin, Kenneth S. McGraw-Hill, 2007. [11] http:/ / jeb. biologists. org/ cgi/ reprint/ 129/ 1/ 141. pdf [13] Viscosity of Welan Gum vs. Concentration in Water. http:/ / www. xydatasource. com/ xy-showdatasetpage. php?datasetcode=345115& dsid=80 [14] http:/ / www. xydatasource. com/ xy-showdatasetpage. php?datasetcode=45615& dsid=76& searchtext=polysaccharide

External links • Polysaccharide Structure (http://employees.csbsju.edu/hjakubowski/classes/ch331/cho/complexoligosacch. htm) • Applications and commercial sources of polysaccharides (http://www.polysaccharidecenter.com) • European Polysaccharide Network of Excellence (http://www.epnoe.eu)

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Metabolism Overview of metabolism Metabolism (from Greek: μεταβολή metabolē, "change" or Greek: μεταβολισμός metabolismos, "outthrow") is the set of life-sustaining chemical transformations within the cells of living organisms. These enzyme-catalyzed reactions allow organisms to grow and reproduce, maintain their structures, and respond to their environments. The word metabolism can also refer to all chemical reactions that occur in living organisms, including digestion and the transport of substances into and between different cells, in which case the set of reactions within the cells is called intermediary metabolism or intermediate metabolism.

Structure of adenosine triphosphate (ATP), a central intermediate in energy metabolism

Metabolism is usually divided into two categories. Catabolism breaks down organic matter, for example to harvest energy in cellular respiration. Anabolism uses energy to construct components of cells such as proteins and nucleic acids. The chemical reactions of metabolism are organized into metabolic pathways, in which one chemical is transformed through a series of steps into another chemical, by a sequence of enzymes. Enzymes are crucial to metabolism because they allow organisms to drive desirable reactions that require energy and will not occur by themselves, by coupling them to spontaneous reactions that release energy. As enzymes act as catalysts they allow these reactions to proceed quickly and efficiently. Enzymes also allow the regulation of metabolic pathways in response to changes in the cell's environment or signals from other cells. The metabolism of an organism determines which substances it will find nutritious and which it will find poisonous. For example, some prokaryotes use hydrogen sulfide as a nutrient, yet this gas is poisonous to animals.[] The speed of metabolism, the metabolic rate, influences how much food an organism will require, and also affects how it is able to obtain that food. A striking feature of metabolism is the similarity of the basic metabolic pathways and components between even vastly different species.[1] For example, the set of carboxylic acids that are best known as the intermediates in the citric acid cycle are present in all known organisms, being found in species as diverse as the unicellular bacterium Escherichia coli and huge multicellular organisms like elephants.[] These striking similarities in metabolic pathways are likely due to their early appearance in evolutionary history, and being retained because of their efficacy.[][]

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Key biochemicals Most of the structures that make up animals, plants and microbes are made from three basic classes of molecule: amino acids, carbohydrates and lipids (often called fats). As these molecules are vital for life, metabolic reactions either focus on making these molecules during the construction of cells and tissues, or breaking them down and using them as a source of energy, in the digestion and use of food. Many important biochemicals can be joined together to make polymers such as DNA and proteins. These macromolecules are essential.

Structure of a triacylglycerol lipid

Type of molecule Name of monomer forms

Name of polymer forms

Examples of polymer forms

Amino acids

Proteins (also called polypeptides) Fibrous proteins and globular proteins

Carbohydrates

Monosaccharides

Polysaccharides

Starch, glycogen and cellulose

Nucleic acids

Nucleotides

Polynucleotides

DNA and RNA

Amino acids and proteins Proteins are made of amino acids arranged in a linear chain and joined together by peptide bonds. Many proteins are the enzymes that catalyze the chemical reactions in metabolism. Other proteins have structural or mechanical functions, such as the proteins that form the cytoskeleton, a system of scaffolding that maintains the cell shape.[2] Proteins are also important in cell signaling, immune responses, cell adhesion, active transport across membranes, and the cell cycle.[] Amino acids also contribute to cellular energy metabolism by providing a carbon source for entry into the tricarboxylic acid cycle,[3] especially a when primary source of energy, such as glucose, is scarce, or when cells undergo metabolic stress.[4]

Lipids Lipids are the most diverse group of biochemicals. Their main structural uses are as part of biological membranes such as the cell membrane, or as a source of energy.[] Lipids are usually defined as hydrophobic or amphipathic biological molecules that will dissolve in organic solvents such as benzene or chloroform.[5] The fats are a large group of compounds that contain fatty acids and glycerol; a glycerol molecule attached to three fatty acid esters is a triacylglyceride.[6] Several variations on this basic structure exist, including alternate backbones such as sphingosine in the sphingolipids, and hydrophilic groups such as phosphate in phospholipids. Steroids such as cholesterol are another major class of lipids that are made in cells.[7]

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Carbohydrates Carbohydrates are aldehydes or ketones with many hydroxyl groups that can exist as straight chains or rings. Carbohydrates are the most abundant biological molecules, and fill numerous roles, such as the storage and transport of energy (starch, glycogen) and structural components (cellulose in plants, chitin in animals).[] The basic carbohydrate units are called monosaccharides and include galactose, fructose, and most importantly glucose. Monosaccharides can be linked together to form polysaccharides in almost limitless ways.[8]

Nucleotides

Glucose can exist in both a straight-chain and ring form.

The two nucleic acids, DNA and RNA are polymers of nucleotides, each nucleotide comprising a phosphate group, a ribose sugar group, and a nitrogenous base. Nucleic acids are critical for the storage and use of genetic information, through the processes of transcription and protein biosynthesis.[] This information is protected by DNA repair mechanisms and propagated through DNA replication. Many viruses have an RNA genome, for example HIV, which uses reverse transcription to create a DNA template from its viral RNA genome.[9] RNA in ribozymes such as spliceosomes and ribosomes is similar to enzymes as it can catalyze chemical reactions. Individual nucleosides are made by attaching a nucleobase to a ribose sugar. These bases are heterocyclic rings containing nitrogen, classified as purines or pyrimidines. Nucleotides also act as coenzymes in metabolic group transfer reactions.[]

Coenzymes Metabolism involves a vast array of chemical reactions, but most fall under a few basic types of reactions that involve the transfer of functional groups.[10] This common chemistry allows cells to use a small set of metabolic intermediates to carry chemical groups between different [] reactions. These group-transfer Structure of the coenzyme acetyl-CoA.The transferable acetyl group is bonded to the sulfur atom at the extreme left. intermediates are called coenzymes. Each class of group-transfer reactions is carried out by a particular coenzyme, which is the substrate for a set of enzymes that produce it, and a set of enzymes that consume it. These coenzymes are therefore continuously being made, consumed and then recycled.[] One central coenzyme is adenosine triphosphate (ATP), the universal energy currency of cells. This nucleotide is used to transfer chemical energy between different chemical reactions. There is only a small amount of ATP in cells, but as it is continuously regenerated, the human body can use about its own weight in ATP per day.[] ATP acts as a bridge between catabolism and anabolism, with catabolic reactions generating ATP and anabolic reactions consuming it. It also serves as a carrier of phosphate groups in phosphorylation reactions. A vitamin is an organic compound needed in small quantities that cannot be made in the cells. In human nutrition, most vitamins function as coenzymes after modification; for example, all water-soluble vitamins are phosphorylated

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or are coupled to nucleotides when they are used in cells.[11] Nicotinamide adenine dinucleotide (NADH), a derivative of vitamin B3 (niacin), is an important coenzyme that acts as a hydrogen acceptor. Hundreds of separate types of dehydrogenases remove electrons from their substrates and reduce NAD+ into NADH. This reduced form of the coenzyme is then a substrate for any of the reductases in the cell that need to reduce their substrates.[12] Nicotinamide adenine dinucleotide exists in two related forms in the cell, NADH and NADPH. The NAD+/NADH form is more important in catabolic reactions, while NADP+/NADPH is used in anabolic reactions.

Minerals and cofactors Inorganic elements play critical roles in metabolism; some are abundant (e.g. sodium and potassium) while others function at minute concentrations. About 99% of a mammal's mass is made up of the elements carbon, nitrogen, calcium, sodium, chlorine, potassium, hydrogen, phosphorus, oxygen and sulfur.[] Organic compounds (proteins, lipids and carbohydrates) contain the majority of the carbon and nitrogen; most of the oxygen and hydrogen is present as water.[] The abundant inorganic elements act as ionic electrolytes. The most important ions are sodium, potassium, calcium, magnesium, chloride, phosphate and the organic ion bicarbonate. The maintenance of Structure of hemoglobin. The protein subunits are in red and blue, and the [1] precise gradients across cell membranes iron-containing heme groups in green. From PDB 1GZX . maintains osmotic pressure and pH.[13] Ions are also critical for nerve and muscle function, as action potentials in these tissues are produced by the exchange of electrolytes between the extracellular fluid and the cytosol.[14] Electrolytes enter and leave cells through proteins in the cell membrane called ion channels. For example, muscle contraction depends upon the movement of calcium, sodium and potassium through ion channels in the cell membrane and T-tubules.[15] Transition metals are usually present as trace elements in organisms, with zinc and iron being most abundant.[16][] These metals are used in some proteins as cofactors and are essential for the activity of enzymes such as catalase and oxygen-carrier proteins such as hemoglobin.[17] Metal cofactors are bound tightly to specific sites in proteins; although enzyme cofactors can be modified during catalysis, they always return to their original state by the end of the reaction catalyzed. Metal micronutrients are taken up into organisms by specific transporters and bind to storage proteins such as ferritin or metallothionein when not being used.[18][19]

Catabolism Catabolism is the set of metabolic processes that break down large molecules. These include breaking down and oxidizing food molecules. The purpose of the catabolic reactions is to provide the energy and components needed by anabolic reactions. The exact nature of these catabolic reactions differ from organism to organism and organisms can be classified based on their sources of energy and carbon (their primary nutritional groups), as shown in the table below. Organic molecules are used as a source of energy by organotrophs, while lithotrophs use inorganic substrates and phototrophs capture sunlight as chemical energy. However, all these different forms of metabolism depend on

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redox reactions that involve the transfer of electrons from reduced donor molecules such as organic molecules, water, ammonia, hydrogen sulfide or ferrous ions to acceptor molecules such as oxygen, nitrate or sulfate.[20] In animals these reactions involve complex organic molecules being broken down to simpler molecules, such as carbon dioxide and water. In photosynthetic organisms such as plants and cyanobacteria, these electron-transfer reactions do not release energy, but are used as a way of storing energy absorbed from sunlight.[]

Classification of organisms based on their metabolism Energy source

sunlight

photo-

-troph

Preformed molecules chemoElectron donor

Carbon source

organic compound

organo-

inorganic compound

litho-

organic compound

hetero-

inorganic compound

auto-

The most common set of catabolic reactions in animals can be separated into three main stages. In the first, large organic molecules such as proteins, polysaccharides or lipids are digested into their smaller components outside cells. Next, these smaller molecules are taken up by cells and converted to yet smaller molecules, usually acetyl coenzyme A (acetyl-CoA), which releases some energy. Finally, the acetyl group on the CoA is oxidised to water and carbon dioxide in the citric acid cycle and electron transport chain, releasing the energy that is stored by reducing the coenzyme nicotinamide adenine dinucleotide (NAD+) into NADH.

Digestion Macromolecules such as starch, cellulose or proteins cannot be rapidly taken up by cells and must be broken into their smaller units before they can be used in cell metabolism. Several common classes of enzymes digest these polymers. These digestive enzymes include proteases that digest proteins into amino acids, as well as glycoside hydrolases that digest polysaccharides into monosaccharides. Microbes simply secrete digestive enzymes into their surroundings,[21][22] while animals only secrete these enzymes from specialized cells in their guts.[23] The amino acids or sugars released by these extracellular enzymes are then pumped into cells by specific active transport proteins.[24][25]

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Energy from organic compounds Carbohydrate catabolism is the breakdown of carbohydrates into smaller units. Carbohydrates are usually taken into cells once they have been digested into monosaccharides.[26] Once inside, the major route of breakdown is glycolysis, where sugars such as glucose and fructose are converted into pyruvate and some ATP is generated.[] Pyruvate is an intermediate in several metabolic pathways, but the majority is converted to acetyl-CoA and fed into the citric acid cycle. Although some more ATP is generated in the citric acid cycle, the most important product is NADH, which is made from NAD+ as the acetyl-CoA is oxidized. This oxidation releases carbon dioxide as a A simplified outline of the catabolism of proteins, carbohydrates and fats waste product. In anaerobic conditions, glycolysis produces lactate, through the enzyme lactate dehydrogenase re-oxidizing NADH to NAD+ for re-use in glycolysis. An alternative route for glucose breakdown is the pentose phosphate pathway, which reduces the coenzyme NADPH and produces pentose sugars such as ribose, the sugar component of nucleic acids. Fats are catabolised by hydrolysis to free fatty acids and glycerol. The glycerol enters glycolysis and the fatty acids are broken down by beta oxidation to release acetyl-CoA, which then is fed into the citric acid cycle. Fatty acids release more energy upon oxidation than carbohydrates because carbohydrates contain more oxygen in their structures. Amino acids are either used to synthesize proteins and other biomolecules, or oxidized to urea and carbon dioxide as a source of energy.[27] The oxidation pathway starts with the removal of the amino group by a transaminase. The amino group is fed into the urea cycle, leaving a deaminated carbon skeleton in the form of a keto acid. Several of these keto acids are intermediates in the citric acid cycle, for example the deamination of glutamate forms α-ketoglutarate.[28] The glucogenic amino acids can also be converted into glucose, through gluconeogenesis (discussed below).[29]

Energy transformations Oxidative phosphorylation In oxidative phosphorylation, the electrons removed from organic molecules in areas such as the protagon acid cycle are transferred to oxygen and the energy released is used to make ATP. This is done in eukaryotes by a series of proteins in the membranes of mitochondria called the electron transport chain. In prokaryotes, these proteins are found in the cell's inner membrane.[30] These proteins use the energy released from passing electrons from reduced molecules like NADH onto oxygen to pump protons across a membrane.[31]

Overview of metabolism

Pumping protons out of the mitochondria creates a proton concentration difference across the membrane and generates an electrochemical gradient.[32] This force drives protons back into the mitochondrion through the base of an enzyme called ATP synthase. The flow of protons makes the stalk subunit rotate, causing the active site of the synthase domain to change shape and phosphorylate adenosine diphosphate– turning it into ATP.[]

Energy from inorganic compounds Chemolithotrophy is a type of metabolism found in prokaryotes where energy is obtained from the oxidation of Mechanism of ATP synthase. ATP is shown in red, ADP and [33] phosphate in pink and the rotating stalk subunit in black. inorganic compounds. These organisms can use hydrogen, reduced sulfur compounds (such as sulfide, hydrogen sulfide and thiosulfate),[] ferrous iron (FeII)[34] or ammonia[35] as sources of reducing power and they gain energy from the oxidation of these compounds with electron acceptors such as oxygen or nitrite.[36] These microbial processes are important in global biogeochemical cycles such as acetogenesis, nitrification and denitrification and are critical for soil fertility.[37][38]

Energy from light The energy in sunlight is captured by plants, cyanobacteria, purple bacteria, green sulfur bacteria and some protists. This process is often coupled to the conversion of carbon dioxide into organic compounds, as part of photosynthesis, which is discussed below. The energy capture and carbon fixation systems can however operate separately in prokaryotes, as purple bacteria and green sulfur bacteria can use sunlight as a source of energy, while switching between carbon fixation and the fermentation of organic compounds.[39][40] In many organisms the capture of solar energy is similar in principle to oxidative phosphorylation, as it involves energy being stored as a proton concentration gradient and this proton motive force then driving ATP synthesis.[] The electrons needed to drive this electron transport chain come from light-gathering proteins called photosynthetic reaction centres or rhodopsins. Reaction centers are classed into two types depending on the type of photosynthetic pigment present, with most photosynthetic bacteria only having one type, while plants and cyanobacteria have two.[41] In plants, algae, and cyanobacteria, photosystem II uses light energy to remove electrons from water, releasing oxygen as a waste product. The electrons then flow to the cytochrome b6f complex, which uses their energy to pump protons across the thylakoid membrane in the chloroplast.[] These protons move back through the membrane as they drive the ATP synthase, as before. The electrons then flow through photosystem I and can then either be used to reduce the coenzyme NADP+, for use in the Calvin cycle, which is discussed below, or recycled for further ATP generation.[42]

Anabolism Anabolism is the set of constructive metabolic processes where the energy released by catabolism is used to synthesize complex molecules. In general, the complex molecules that make up cellular structures are constructed step-by-step from small and simple precursors. Anabolism involves three basic stages. Firstly, the production of precursors such as amino acids, monosaccharides, isoprenoids and nucleotides, secondly, their activation into reactive forms using energy from ATP, and thirdly, the assembly of these precursors into complex molecules such as proteins, polysaccharides, lipids and nucleic acids.

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Organisms differ in how many of the molecules in their cells they can construct for themselves. Autotrophs such as plants can construct the complex organic molecules in cells such as polysaccharides and proteins from simple molecules like carbon dioxide and water. Heterotrophs, on the other hand, require a source of more complex substances, such as monosaccharides and amino acids, to produce these complex molecules. Organisms can be further classified by ultimate source of their energy: photoautotrophs and photoheterotrophs obtain energy from light, whereas chemoautotrophs and chemoheterotrophs obtain energy from inorganic oxidation reactions.

Carbon fixation Photosynthesis is the synthesis of carbohydrates from sunlight and carbon dioxide (CO2). In plants, cyanobacteria and algae, oxygenic photosynthesis splits water, with oxygen produced as a waste product. This process uses the ATP and NADPH produced by the photosynthetic reaction centres, as described above, to convert CO2 into glycerate 3-phosphate, which can then be converted into glucose. This carbon-fixation reaction is carried out by the enzyme RuBisCO as part of the Calvin– Benson cycle.[43] Three types of photosynthesis occur in plants, C3 carbon fixation, C4 carbon fixation and CAM photosynthesis. These differ by the route that carbon dioxide takes to the Calvin cycle, with C3 plants fixing CO2 directly, while C4 and CAM photosynthesis incorporate the CO2 into other compounds first, as adaptations to deal with intense sunlight and dry conditions.[44]

Plant cells (bounded by purple walls) filled with chloroplasts (green), which are the site of photosynthesis

In photosynthetic prokaryotes the mechanisms of carbon fixation are more diverse. Here, carbon dioxide can be fixed by the Calvin– Benson cycle, a reversed citric acid cycle,[45] or the carboxylation of acetyl-CoA.[46][47] Prokaryotic chemoautotrophs also fix CO2 through the Calvin– Benson cycle, but use energy from inorganic compounds to drive the reaction.[48]

Carbohydrates and glycans In carbohydrate anabolism, simple organic acids can be converted into monosaccharides such as glucose and then used to assemble polysaccharides such as starch. The generation of glucose from compounds like pyruvate, lactate, glycerol, glycerate 3-phosphate and amino acids is called gluconeogenesis. Gluconeogenesis converts pyruvate to glucose-6-phosphate through a series of intermediates, many of which are shared with glycolysis.[] However, this pathway is not simply glycolysis run in reverse, as several steps are catalyzed by non-glycolytic enzymes. This is important as it allows the formation and breakdown of glucose to be regulated separately, and prevents both pathways from running simultaneously in a futile cycle.[49][50] Although fat is a common way of storing energy, in vertebrates such as humans the fatty acids in these stores cannot be converted to glucose through gluconeogenesis as these organisms cannot convert acetyl-CoA into pyruvate; plants do, but animals do not, have the necessary enzymatic machinery.[] As a result, after long-term starvation, vertebrates need to produce ketone bodies from fatty acids to replace glucose in tissues such as the brain that cannot metabolize fatty acids.[51] In other organisms such as plants and bacteria, this metabolic problem is solved using the glyoxylate cycle, which bypasses the decarboxylation step in the citric acid cycle and allows the transformation of acetyl-CoA to oxaloacetate, where it can be used for the production of glucose.[][] Polysaccharides and glycans are made by the sequential addition of monosaccharides by glycosyltransferase from a reactive sugar-phosphate donor such as uridine diphosphate glucose (UDP-glucose) to an acceptor hydroxyl group on the growing polysaccharide. As any of the hydroxyl groups on the ring of the substrate can be acceptors, the polysaccharides produced can have straight or branched structures.[52] The polysaccharides produced can have structural or metabolic functions themselves, or be transferred to lipids and proteins by enzymes called

Overview of metabolism oligosaccharyltransferases.[53][54]

Fatty acids, isoprenoids and steroids Fatty acids are made by fatty acid synthases that polymerize and then reduce acetyl-CoA units. The acyl chains in the fatty acids are extended by a cycle of reactions that add the acyl group, reduce it to an alcohol, dehydrate it to an alkene group and then reduce it again to an alkane group. The enzymes of fatty acid biosynthesis are divided into two groups, in animals and fungi all these fatty acid synthase reactions are carried out by a single multifunctional type I protein,[55] while in plant plastids and bacteria separate type II enzymes perform each step in the pathway.[56][57] Terpenes and isoprenoids are a large class of lipids that include the carotenoids and form the largest class of plant natural products.[58] These compounds are made by the assembly and modification of isoprene units Simplified version of the steroid synthesis pathway with the intermediates isopentenyl donated from the reactive precursors pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate isopentenyl pyrophosphate and (GPP) and squalene shown. Some intermediates are omitted for clarity. dimethylallyl pyrophosphate.[] These precursors can be made in different ways. In animals and archaea, the mevalonate pathway produces these compounds from acetyl-CoA,[59] while in plants and bacteria the non-mevalonate pathway uses pyruvate and glyceraldehyde 3-phosphate as substrates.[][60] One important reaction that uses these activated isoprene donors is steroid biosynthesis. Here, the isoprene units are joined together to make squalene and then folded up and formed into a set of rings to make lanosterol.[] Lanosterol can then be converted into other steroids such as cholesterol and ergosterol.[][61]

Proteins Organisms vary in their ability to synthesize the 20 common amino acids. Most bacteria and plants can synthesize all twenty, but mammals can only synthesize eleven nonessential amino acids, so nine essential amino acids must be obtained from food.[] Some simple parasites, such as the bacteria Mycoplasma pneumoniae, lack all amino acid synthesis and take their amino acids directly from their hosts.[62] All amino acids are synthesized from intermediates in glycolysis, the citric acid cycle, or the pentose phosphate pathway. Nitrogen is provided by glutamate and glutamine. Amino acid synthesis depends on the formation of the appropriate alpha-keto acid, which is then transaminated to form an amino acid.[63] Amino acids are made into proteins by being joined together in a chain by peptide bonds. Each different protein has a unique sequence of amino acid residues: this is its primary structure. Just as the letters of the alphabet can be

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Overview of metabolism combined to form an almost endless variety of words, amino acids can be linked in varying sequences to form a huge variety of proteins. Proteins are made from amino acids that have been activated by attachment to a transfer RNA molecule through an ester bond. This aminoacyl-tRNA precursor is produced in an ATP-dependent reaction carried out by an aminoacyl tRNA synthetase.[64] This aminoacyl-tRNA is then a substrate for the ribosome, which joins the amino acid onto the elongating protein chain, using the sequence information in a messenger RNA.[65]

Nucleotide synthesis and salvage Nucleotides are made from amino acids, carbon dioxide and formic acid in pathways that require large amounts of metabolic energy.[66] Consequently, most organisms have efficient systems to salvage preformed nucleotides.[66][67] Purines are synthesized as nucleosides (bases attached to ribose). Both adenine and guanine are made from the precursor nucleoside inosine monophosphate, which is synthesized using atoms from the amino acids glycine, glutamine, and aspartic acid, as well as formate transferred from the coenzyme tetrahydrofolate. Pyrimidines, on the other hand, are synthesized from the base orotate, which is formed from glutamine and aspartate.[68]

Xenobiotics and redox metabolism All organisms are constantly exposed to compounds that they cannot use as foods and would be harmful if they accumulated in cells, as they have no metabolic function. These potentially damaging compounds are called xenobiotics.[69] Xenobiotics such as synthetic drugs, natural poisons and antibiotics are detoxified by a set of xenobiotic-metabolizing enzymes. In humans, these include cytochrome P450 oxidases,[70] UDP-glucuronosyltransferases,[71] and glutathione S-transferases.[72] This system of enzymes acts in three stages to firstly oxidize the xenobiotic (phase I) and then conjugate water-soluble groups onto the molecule (phase II). The modified water-soluble xenobiotic can then be pumped out of cells and in multicellular organisms may be further metabolized before being excreted (phase III). In ecology, these reactions are particularly important in microbial biodegradation of pollutants and the bioremediation of contaminated land and oil spills.[73] Many of these microbial reactions are shared with multicellular organisms, but due to the incredible diversity of types of microbes these organisms are able to deal with a far wider range of xenobiotics than multicellular organisms, and can degrade even persistent organic pollutants such as organochloride compounds.[74] A related problem for aerobic organisms is oxidative stress.[] Here, processes including oxidative phosphorylation and the formation of disulfide bonds during protein folding produce reactive oxygen species such as hydrogen peroxide.[75] These damaging oxidants are removed by antioxidant metabolites such as glutathione and enzymes such as catalases and peroxidases.[][]

Thermodynamics of living organisms Living organisms must obey the laws of thermodynamics, which describe the transfer of heat and work. The second law of thermodynamics states that in any closed system, the amount of entropy (disorder) will tend to increase. Although living organisms' amazing complexity appears to contradict this law, life is possible as all organisms are open systems that exchange matter and energy with their surroundings. Thus living systems are not in equilibrium, but instead are dissipative systems that maintain their state of high complexity by causing a larger increase in the entropy of their environments.[76] The metabolism of a cell achieves this by coupling the spontaneous processes of catabolism to the non-spontaneous processes of anabolism. In thermodynamic terms, metabolism maintains order by creating disorder.[77]

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Regulation and control As the environments of most organisms are constantly changing, the reactions of metabolism must be finely regulated to maintain a constant set of conditions within cells, a condition called homeostasis.[78][79] Metabolic regulation also allows organisms to respond to signals and interact actively with their environments.[80] Two closely linked concepts are important for understanding how metabolic pathways are controlled. Firstly, the regulation of an enzyme in a pathway is how its activity is increased and decreased in response to signals. Secondly, the control exerted by this enzyme is the effect that these changes in its activity have on the overall rate of the pathway (the flux through the pathway).[] For example, an enzyme may show large changes in activity (i.e. it is highly regulated) but if these changes have little effect on the flux of a metabolic pathway, then this enzyme is not involved in the control of the pathway.[81] There are multiple levels of metabolic regulation. In intrinsic regulation, the metabolic pathway self-regulates to respond to changes in the levels of substrates or products; for example, a decrease in the amount of product can increase the flux through the pathway to compensate.[] This type of regulation often involves allosteric regulation of the activities of multiple enzymes in the pathway.[82] Extrinsic control involves a cell in a multicellular Effect of insulin on glucose uptake and metabolism. Insulin binds to its receptor organism changing its metabolism in (1), which in turn starts many protein activation cascades (2). These include: response to signals from other cells. These translocation of Glut-4 transporter to the plasma membrane and influx of glucose (3), glycogen synthesis (4), glycolysis (5) and fatty acid synthesis (6). signals are usually in the form of soluble messengers such as hormones and growth factors and are detected by specific receptors on the cell surface.[83] These signals are then transmitted inside the cell by second messenger systems that often involved the phosphorylation of proteins.[84] A very well understood example of extrinsic control is the regulation of glucose metabolism by the hormone insulin.[85] Insulin is produced in response to rises in blood glucose levels. Binding of the hormone to insulin receptors on cells then activates a cascade of protein kinases that cause the cells to take up glucose and convert it into storage molecules such as fatty acids and glycogen.[86] The metabolism of glycogen is controlled by activity of phosphorylase, the enzyme that breaks down glycogen, and glycogen synthase, the enzyme that makes it. These enzymes are regulated in a reciprocal fashion, with phosphorylation inhibiting glycogen synthase, but activating phosphorylase. Insulin causes glycogen synthesis by activating protein phosphatases and producing a decrease in the phosphorylation of these enzymes.[87]

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Evolution The central pathways of metabolism described above, such as glycolysis and the citric acid cycle, are present in all three domains of living things and were present in the last universal ancestor.[][88] This universal ancestral cell was prokaryotic and probably a methanogen that had extensive amino acid, nucleotide, carbohydrate and lipid metabolism.[89][90] The retention of these ancient pathways during later evolution may be the result of these reactions being an optimal solution to their particular metabolic problems, with pathways such as glycolysis and Evolutionary tree showing the common ancestry of organisms from all three domains of life. Bacteria are colored blue, eukaryotes red, and archaea green. Relative positions of the citric acid cycle producing their some of the phyla included are shown around the tree. end products highly efficiently and in a minimal number of steps.[][] Mutation changes that affect non-coding DNA segments may merely affect the metabolic efficiency of the individual for whom the mutation occurs.[91] The first pathways of enzyme-based metabolism may have been parts of purine nucleotide metabolism, with previous metabolic pathways being part of the ancient RNA world.[92] Many models have been proposed to describe the mechanisms by which novel metabolic pathways evolve. These include the sequential addition of novel enzymes to a short ancestral pathway, the duplication and then divergence of entire pathways as well as the recruitment of pre-existing enzymes and their assembly into a novel reaction pathway.[93] The relative importance of these mechanisms is unclear, but genomic studies have shown that enzymes in a pathway are likely to have a shared ancestry, suggesting that many pathways have evolved in a step-by-step fashion with novel functions being created from pre-existing steps in the pathway.[94] An alternative model comes from studies that trace the evolution of proteins' structures in metabolic networks, this has suggested that enzymes are pervasively recruited, borrowing enzymes to perform similar functions in different metabolic pathways (evident in the MANET database)[95] These recruitment processes result in an evolutionary enzymatic mosaic.[96] A third possibility is that some parts of metabolism might exist as "modules" that can be reused in different pathways and perform similar functions on different molecules.[97] As well as the evolution of new metabolic pathways, evolution can also cause the loss of metabolic functions. For example, in some parasites metabolic processes that are not essential for survival are lost and preformed amino acids, nucleotides and carbohydrates may instead be scavenged from the host.[98] Similar reduced metabolic capabilities are seen in endosymbiotic organisms.[99]

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Investigation and manipulation Classically, metabolism is studied by a reductionist approach that focuses on a single metabolic pathway. Particularly valuable is the use of radioactive tracers at the whole-organism, tissue and cellular levels, which define the paths from precursors to final products by identifying radioactively labelled intermediates and products.[100] The enzymes that catalyze these chemical reactions can then be purified and their kinetics and responses to inhibitors investigated. A parallel approach is to identify the small molecules in a cell or tissue; the complete set of these molecules is called the metabolome. Overall, these studies give a good view of the structure and function of simple metabolic pathways, but are inadequate when applied to more complex systems such as the metabolism of a complete cell.[101]

Metabolic network of the Arabidopsis thaliana citric acid cycle. Enzymes and metabolites are shown as red squares and the interactions between them as black lines.

An idea of the complexity of the metabolic networks in cells that contain thousands of different enzymes is given by the figure showing the interactions between just 43 proteins and 40 metabolites to the right: the sequences of genomes provide lists containing anything up to 45,000 genes.[102] However, it is now possible to use this genomic data to reconstruct complete networks of biochemical reactions and produce more holistic mathematical models that may explain and predict their behavior.[103] These models are especially powerful when used to integrate the pathway and metabolite data obtained through classical methods with data on gene expression from proteomic and DNA microarray studies.[104] Using these techniques, a model of human metabolism has now been produced, which will guide future drug discovery and biochemical research.[105] These models are now being used in network analysis, to classify human diseases into groups that share common proteins or metabolites.[106][107] Bacterial metabolic networks are a striking example of bow-tie[][][] organization, an architecture able to input a wide range of nutrients and produce a large variety of products and complex macromolecules using a relatively few intermediate common currencies. A major technological application of this information is metabolic engineering. Here, organisms such as yeast, plants or bacteria are genetically modified to make them more useful in biotechnology and aid the production of drugs such as antibiotics or industrial chemicals such as 1,3-propanediol and shikimic acid.[108] These genetic modifications usually aim to reduce the amount of energy used to produce the product, increase yields and reduce the production of wastes.[109]

Overview of metabolism

History The term metabolism is derived from the Greek Μεταβολισμός– "Metabolismos" for "change", or "overthrow".[110] The history of the scientific study of metabolism spans several centuries and has moved from examining whole animals in early studies, to examining individual metabolic reactions in modern biochemistry. The first controlled experiments in human metabolism were published by Santorio Santorio in 1614 in his book Ars de statica medicina.[111] He described how he weighed himself before and after eating, sleep, working, sex, fasting, drinking, and excreting. He found that most of the food he took in was lost through what he called "insensible perspiration". In these early studies, the mechanisms of these metabolic processes had not been identified and a vital force was thought to animate living tissue.[112] In the 19th century, when studying the fermentation of sugar to alcohol by yeast, Louis Pasteur concluded that fermentation was catalyzed by substances Santorio Santorio in his steelyard within the yeast cells he called "ferments". He wrote that "alcoholic balance, from Ars de statica medicina, fermentation is an act correlated with the life and organization of the yeast first published 1614 cells, not with the death or putrefaction of the cells."[113] This discovery, along with the publication by Friedrich Wöhler in 1828 of the chemical synthesis of urea,[114] notable for being the first organic compound prepared from wholly inorganic precursors, proved that the organic compounds and chemical reactions found in cells were no different in principle than any other part of chemistry. It was the discovery of enzymes at the beginning of the 20th century by Eduard Buchner that separated the study of the chemical reactions of metabolism from the biological study of cells, and marked the beginnings of biochemistry.[115] The mass of biochemical knowledge grew rapidly throughout the early 20th century. One of the most prolific of these modern biochemists was Hans Krebs who made huge contributions to the study of metabolism.[116] He discovered the urea cycle and later, working with Hans Kornberg, the citric acid cycle and the glyoxylate cycle.[117][] Modern biochemical research has been greatly aided by the development of new techniques such as chromatography, X-ray diffraction, NMR spectroscopy, radioisotopic labelling, electron microscopy and molecular dynamics simulations. These techniques have allowed the discovery and detailed analysis of the many molecules and metabolic pathways in cells.

References [91] C.Michael Hogan. 2010. Mutation. ed. E.Monosson and C.J.Cleveland. Encyclopedia of Earth. National Council for Science and the Environment. Washington DC (http:/ / www. eoearth. org/ article/ Mutation?topic=49496) [112] Williams, H. S. (1904) A History of Science: in Five Volumes. Volume IV: Modern Development of the Chemical and Biological Sciences (http:/ / etext. lib. virginia. edu/ toc/ modeng/ public/ Wil4Sci. html) Harper and Brothers (New York) Retrieved on 2007-03-26 [115] Eduard Buchner's 1907 Nobel lecture (http:/ / nobelprize. org/ nobel_prizes/ chemistry/ laureates/ 1907/ buchner-lecture. html) at http:/ / nobelprize. org Accessed 2007-03-20

Further reading • Books about Metabolism: Online books (http://onlinebooks.library.upenn.edu/webbin/ftl?st=&su=Metabolism&library=OLBP), Resources in your library (http://onlinebooks.library.upenn.edu/webbin/ftl?st=&su=Metabolism),

198

Overview of metabolism Resources in other libraries (http:/ / onlinebooks. library. upenn. edu/ webbin/ ftl?st=& su=Metabolism& library=0CHOOSE0) Introductory • Rose, S. and Mileusnic, R., The Chemistry of Life. (Penguin Press Science, 1999), ISBN 0-14-027273-9 • Schneider, E. D. and Sagan, D., Into the Cool: Energy Flow, Thermodynamics, and Life. (University Of Chicago Press, 2005), ISBN 0-226-73936-8 • Lane, N., Oxygen: The Molecule that Made the World. (Oxford University Press, USA, 2004), ISBN 0-19-860783-0 Advanced • Price, N. and Stevens, L., Fundamentals of Enzymology: Cell and Molecular Biology of Catalytic Proteins. (Oxford University Press, 1999), ISBN 0-19-850229-X • Berg, J. Tymoczko, J. and Stryer, L., Biochemistry. (W. H. Freeman and Company, 2002), ISBN 0-7167-4955-6 • Cox, M. and Nelson, D. L., Lehninger Principles of Biochemistry. (Palgrave Macmillan, 2004), ISBN 0-7167-4339-6 • Brock, T. D. Madigan, M. T. Martinko, J. and Parker J., Brock's Biology of Microorganisms. (Benjamin Cummings, 2002), ISBN 0-13-066271-2 • Da Silva, J.J.R.F. and Williams, R. J. P., The Biological Chemistry of the Elements: The Inorganic Chemistry of Life. (Clarendon Press, 1991), ISBN 0-19-855598-9 • Nicholls, D. G. and Ferguson, S. J., Bioenergetics. (Academic Press Inc., 2002), ISBN 0-12-518121-3

External links • biochemical families: carbohydrates • alcohols • glycoproteins • glycosides • lipids • • • • •

eicosanoids fatty acids / intermediates phospholipids sphingolipids steroids

• nucleic acids • constituents / intermediates • proteins • amino acids / intermediates • tetrapyrroles / intermediates

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200

Carbohydrate metabolism Glycolysis Glycolysis (from glycose, an older term[1] for glucose + -lysis degradation) is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+. The free energy released in this process is used to form the high-energy compounds ATP (adenosine triphosphate) and NADH (reduced nicotinamide adenine dinucleotide).[2] Glycolysis overview

Glycolysis is a determined sequence of ten reactions involving ten intermediate compounds (one of the steps involves two intermediates). The intermediates provide entry points to glycolysis. For example, most monosaccharides, such as fructose, glucose, and galactose, can be converted to one of these intermediates. The intermediates may also be directly useful. For example, the intermediate dihydroxyacetone phosphate (DHAP) is a source of the glycerol that combines with fatty acids to form fat. It occurs, with variations, in nearly all organisms, both aerobic and anaerobic. The wide occurrence of glycolysis indicates that it is one of the most ancient known metabolic pathways.[3] It occurs in the cytosol of the cell. The most common type of glycolysis is the Embden–Meyerhof–Parnas (EMP pathway), which was first discovered by Gustav Embden, Otto Meyerhof, and Jakub Karol Parnas. Glycolysis also refers to other pathways, such as the Entner–Doudoroff pathway and various heterofermentative and homofermentative pathways. However, the discussion here will be limited to the Embden–Meyerhof–Parnas pathway.[4] The entire glycolysis pathway can be separated into two phases:[5] 1. The Preparatory Phase – in which ATP is consumed and is hence also known as the investment phase 2. The Pay Off Phase – in which ATP is produced.

Overview The overall reaction of glycolysis is: D-[Glucose]

[Pyruvate] + 2 [NAD]+ + 2 [ADP] + 2 [P]i

+ 2 [NADH] + 2 H+ + 2 [ATP] + 2 H2O

The use of symbols in this equation makes it appear unbalanced with respect to oxygen atoms, hydrogen atoms, and charges. Atom balance is maintained by the two phosphate (Pi) groups:[] • each exists in the form of a hydrogen phosphate anion (HPO42−), dissociating to contribute 2 H+ overall

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201

• each liberates an oxygen atom when it binds to an ADP (adenosine diphosphate) molecule, contributing 2 O overall Charges are balanced by the difference between ADP and ATP. In the cellular environment, all three hydroxy groups of ADP dissociate into −O- and H+, giving ADP3−, and this ion tends to exist in an ionic bond with Mg2+, giving ADPMg-. ATP behaves identically except that it has four hydroxy groups, giving ATPMg2−. When these differences along with the true charges on the two phosphate groups are considered together, the net charges of −4 on each side are balanced. For simple fermentations, the metabolism of one molecule of glucose to two molecules of pyruvate has a net yield of two molecules of ATP. Most cells will then carry out further reactions to 'repay' the used NAD+ and produce a final product of ethanol or lactic acid. Many bacteria use inorganic compounds as hydrogen acceptors to regenerate the NAD+. Cells performing aerobic respiration synthesize much more ATP, but not as part of glycolysis. These further aerobic reactions use pyruvate and NADH + H+ from glycolysis. Eukaryotic aerobic respiration produces approximately 34 additional molecules of ATP for each glucose molecule, however most of these are produced by a vastly different mechanism to the substrate-level phosphorylation in glycolysis. Glycolysis

The lower-energy production, per glucose, of anaerobic respiration relative to aerobic respiration, results in greater flux through the pathway under hypoxic (low-oxygen) conditions, unless alternative sources of anaerobically oxidizable substrates, such as fatty acids, are found. Metabolism of common monosaccharides, including glycolysis, gluconeogenesis, glycogenesis and glycogenolysis

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202

Elucidation of the pathway In 1860, Louis Pasteur discovered that microorganisms are responsible for fermentation. In 1897, Eduard Buchner found that extracts of certain cells can cause fermentation. In 1905, Arthur Harden and William Young along with Nick Sheppard determined that a heat-sensitive high-molecular-weight subcellular fraction (the enzymes) and a heat-insensitive low-molecular-weight cytoplasm fraction (ADP, ATP and NAD+ and other cofactors) are required together for fermentation to proceed. The details of the pathway were eventually determined by 1940, with a major input from Otto Meyerhof and some years later by Luis Leloir. The biggest difficulties in determining the intricacies of the pathway were due to the very short lifetime and low steady-state concentrations of the intermediates of the fast glycolytic reactions.

Sequence of reactions Preparatory phase The first five steps are regarded as the preparatory (or investment) phase, since they consume energy to convert the glucose into two three-carbon sugar phosphates[5] (G3P). The first step in glycolysis is phosphorylation of glucose by a family of enzymes called hexokinases to form glucose 6-phosphate (G6P). This reaction consumes ATP, but it acts to keep the glucose concentration low, promoting continuous transport of glucose into the cell through the plasma membrane transporters. In addition, it blocks the glucose from leaking out – the cell lacks transporters for G6P, and free diffusion out of the cell is prevented due to the charged nature of G6P. Glucose may alternatively be formed from the phosphorolysis or hydrolysis of intracellular starch or glycogen.

D-Glucose (Glc)

In animals, an isozyme of hexokinase called glucokinase is also used in the liver, which has a much lower affinity for glucose (Km in the vicinity of normal glycemia), and differs in regulatory properties. The different substrate affinity and alternate regulation of this enzyme are a reflection of the role of the liver in maintaining blood sugar levels.

Hexokinase (HK) a transferase

α-D-Glucose-6-phosphate (G6P)

ATP H+ + ADP

Cofactors: Mg2+

G6P is then rearranged into fructose 6-phosphate (F6P) by glucose phosphate isomerase. Fructose can also enter the glycolytic pathway by phosphorylation at this point. The change in structure is an isomerization, in which the G6P has been converted to F6P. The reaction requires an enzyme, phosphohexose isomerase, to proceed. This reaction is freely reversible under normal cell conditions. However, it is often driven forward because of a low concentration of F6P, which is constantly consumed during the next step of glycolysis. Under conditions of high F6P concentration, this reaction readily runs in reverse. This phenomenon can be explained through Le Chatelier's Principle. Isomerization to a keto sugar is necessary for carbanion stabilization in the fourth reaction step (below).

α-D-Glucose 6-phosphate (G6P)

Phosphoglucose isomerase an isomerase

β-D-Fructose 6-phosphate (F6P)

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203

The energy expenditure of another ATP in this step is justified in 2 ways: The glycolytic process (up to this step) is now irreversible, and the energy supplied destabilizes the molecule. Because the reaction catalyzed by Phosphofructokinase 1 (PFK-1) is coupled to the hydrolysis of ATP, an energetically favorable step, it is, in essence, irreversible, and a different pathway must be used to do the reverse conversion during gluconeogenesis. This makes the reaction a key regulatory point (see below). This is also the rate-limiting step.

β-D-Fructose 6-phosphate (F6P)

phosphofructokinase (PFK-1) a transferase

ATP

β-D-Fructose 1,6-bisphosphate (F1,6BP)

H+ + ADP

Furthermore, the second phosphorylation event is necessary to allow the formation of two charged groups (rather than only one) in the subsequent step of glycolysis, ensuring the prevention of free diffusion of substrates out of the cell. The same reaction can also be catalyzed by pyrophosphate-dependent phosphofructokinase (PFP or PPi-PFK), which is found in most plants, some bacteria, archea, and protists, but not in animals. This enzyme uses pyrophosphate (PPi) as a phosphate donor instead of ATP. It is a reversible reaction, [6] increasing the flexibility of glycolytic metabolism. A rarer ADP-dependent PFK enzyme variant has [7] been identified in archaean species. Cofactors: Mg2+

Destabilizing the molecule in the previous reaction allows the hexose ring to be split by aldolase into two triose sugars, dihydroxyacetone phosphate, a ketone, and glyceraldehyde 3-phosphate, an aldehyde. There are two classes of aldolases: class I aldolases, present in animals and plants, and class II aldolases, present in fungi and bacteria; the two classes use different mechanisms in cleaving the ketose ring. Electrons delocalized in the carbon-carbon bond cleavage associate with the alcohol group. The resulting carbanion is stabilized by the structure of the carbanion itself via resonance charge distribution and by the presence of a charged ion prosthetic group.

β-D-Fructose 1,6-bisphosphate (F1,6BP)

fructose-bisphosphate aldolase (ALDO) a lyase

Dihydroxyacetone phosphate (DHAP)

D-glyceraldehyde 3-phosphate (GADP) +

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204

Triosephosphate isomerase rapidly interconverts dihydroxyacetone phosphate with glyceraldehyde 3-phosphate (GADP) that proceeds further into glycolysis. This is advantageous, as it directs dihydroxyacetone phosphate down the same pathway as glyceraldehyde 3-phosphate, simplifying regulation.

Dihydroxyacetone phosphate (DHAP)

triosephosphate isomerase (TPI) an isomerase

D-glyceraldehyde 3-phosphate (GADP)

Pay-off phase The second half of glycolysis is known as the pay-off phase, characterised by a net gain of the energy-rich molecules ATP and NADH.[5] Since glucose leads to two triose sugars in the preparatory phase, each reaction in the pay-off phase occurs twice per glucose molecule. This yields 2 NADH molecules and 4 ATP molecules, leading to a net gain of 2 NADH molecules and 2 ATP molecules from the glycolytic pathway per glucose. The triose sugars are dehydrogenated and inorganic phosphate is added to them, forming 1,3-bisphosphoglycerate. The hydrogen is used to reduce two molecules of NAD+, a hydrogen carrier, to give NADH + H+ for each triose.

glyceraldehyde 3-phosphate (GADP)

Hydrogen atom balance and charge balance are both maintained because the phosphate (Pi) group actually exists in the form of a [] hydrogen phosphate anion (HPO42-), which dissociates to contribute the extra H+ ion and gives a net charge of -3 on both sides.

This step is the enzymatic transfer of a phosphate group from 1,3-bisphosphoglycerate to ADP by phosphoglycerate kinase, forming ATP and 3-phosphoglycerate. At this step, glycolysis has reached the break-even point: 2 molecules of ATP were consumed, and 2 new molecules have now been synthesized. This step, one of the two substrate-level phosphorylation steps, requires ADP; thus, when the cell has plenty of ATP (and little ADP), this reaction does not occur. Because ATP decays relatively quickly when it is not metabolized, this is an important regulatory point in the glycolytic pathway..

glyceraldehyde phosphate dehydrogenase (GAPDH) an oxidoreductase

NAD+ + Pi

1,3-bisphosphoglycerate (1,3-BPG)

D-1,3-bisphosphoglycerate (1,3BPG)

NADH + H+

phosphoglycerate kinase (PGK) a transferase

ADP

ATP

ADP actually exists as ADPMg-, and ATP as ATPMg2-, balancing the charges at -5 both sides. Cofactors: Mg2+ phosphoglycerate kinase (PGK)

3-phosphoglycerate (3-P-G)

Glycolysis

Phosphoglycerate mutase now forms 2-phosphoglycerate.

205

3-phosphoglycerate (3PG)

Enolase next forms phosphoenolpyruvate from 2-phosphoglycerate. Cofactors: 2 Mg2+: one "conformational" ion to coordinate with the carboxylate group of the substrate, and one "catalytic" ion that participates in the dehydration.

phosphoglycerate mutase (PGM) a mutase

2-phosphoglycerate (2PG)

enolase (ENO) a lyase

phosphoenolpyruvate (PEP)

H2O

enolase (ENO)

A final substrate-level phosphorylation now forms a molecule of pyruvate and a molecule of ATP by means of the enzyme pyruvate kinase. This serves as an additional regulatory step, similar to the phosphoglycerate kinase step. Cofactors: Mg2+

phosphoenolpyruvate (PEP)

pyruvate kinase (PK) a transferase

ADP + H+

pyruvate (Pyr)

ATP

Regulation Glycolysis is regulated by slowing down or speeding up certain steps in the glycolysis pathway. This is accomplished by inhibiting or activating the enzymes that are involved. The steps that are regulated may be determined by calculating the change in free energy, ΔG, for each step. If a step's products and reactants are in equilibrium, then the step is assumed not to be regulated. Since the change in free energy is zero for a system at equilibrium, any step with a free energy change near zero is not being regulated. If a step is being regulated, then that step's enzyme is not converting reactants into products as fast as it could, resulting in a build-up of reactants, which would be converted to products if the enzyme were operating faster. Since the reaction is thermodynamically favorable, the change in free energy for the step will be negative. A step with a large negative change in free energy is assumed to be regulated.

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206

Free energy changes Concentrations of metabolites in erythrocytes[8] Compound

Concentration / mM

glucose

5.0

glucose-6-phosphate

0.083

fructose-6-phosphate

0.014

fructose-1,6-bisphosphate

0.031

dihydroxyacetone phosphate

0.14

glyceraldehyde-3-phosphate

0.019

1,3-bisphosphoglycerate

0.001

2,3-bisphosphoglycerate

4.0

3-phosphoglycerate

0.12

2-phosphoglycerate

0.03

phosphoenolpyruvate

0.023

pyruvate

0.051

ATP

1.85

ADP

0.14

1.0

The change in free energy for each step of glycolysis estimated from the concentration of metabolites in an erythrocyte.

The change in free energy, ΔG, for each step in the glycolysis pathway can be calculated using ΔG = ΔG°' + RTln Q, where Q is the reaction quotient. This requires knowing the concentrations of the metabolites. All of these values are available for erythrocytes, with the exception of the concentrations of NAD+ and NADH. The ratio of NAD+ to NADH in the cytoplasm is approximately 1000, which makes the oxidation of glyceraldehyde-3-phosphate (step 6) more favourable. Using the measured concentrations of each step, and the standard free energy changes, the actual free energy change can be calculated. (Neglecting this is very common - the delta G of ATP hydrolysis in cells is not the standard free energy change of ATP hydrolysis quoted in textbooks).

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207

Change in free energy for each step of glycolysis[9] Step

Reaction

ΔG°' / (kJ/mol) ΔG / (kJ/mol)

glucose + ATP4- → glucose-6-phosphate2- + ADP3- + H+

-16.7

-34

glucose-6-phosphate2- → fructose-6-phosphate2-

1.67

-2.9

fructose-6-phosphate2- + ATP4- → fructose-1,6-bisphosphate4- + ADP3- + H+

-14.2

-19

fructose-1,6-bisphosphate4- → dihydroxyacetone phosphate2- + glyceraldehyde-3-phosphate2- 23.9

dihydroxyacetone phosphate2- → glyceraldehyde-3-phosphate2-

7.56

2.4

glyceraldehyde-3-phosphate2- + Pi2- + NAD+ → 1,3-bisphosphoglycerate4- + NADH + H+

6.30

-1.29

1,3-bisphosphoglycerate4- + ADP3- → 3-phosphoglycerate3- + ATP4-

-18.9

0.09

3-phosphoglycerate3- → 2-phosphoglycerate3-

4.4

0.83

2-phosphoglycerate3- → phosphoenolpyruvate3- + H2O

1.8

1.1

phosphoenolpyruvate3- + ADP3- + H+ → pyruvate- + ATP4-

-31.7

-23.0

-0.23

From measuring the physiological concentrations of metabolites in an erythrocyte it seems that about seven of the steps in glycolysis are in equilibrium for that cell type. Three of the steps — the ones with large negative free energy changes — are not in equilibrium and are referred to as irreversible; such steps are often subject to regulation. Step 5 in the figure is shown behind the other steps, because that step is a side-reaction that can decrease or increase the concentration of the intermediate glyceraldehyde-3-phosphate. That compound is converted to dihydroxyacetone phosphate by the enzyme triose phosphate isomerase, which is a catalytically perfect enzyme; its rate is so fast that the reaction can be assumed to be in equilibrium. The fact that ΔG is not zero indicates that the actual concentrations in the erythrocyte are not accurately known.

Biochemical logic The existence of more than one point of regulation indicates that intermediates between those points enter and leave the glycolysis pathway by other processes. For example, in the first regulated step, hexokinase converts glucose into glucose-6-phosphate. Instead of continuing through the glycolysis pathway, this intermediate can be converted into glucose storage molecules, such as glycogen or starch. The reverse reaction, breaking down, e.g., glycogen, produces mainly glucose-6-phosphate; very little free glucose is formed in the reaction. The glucose-6-phosphate so produced can enter glycolysis after the first control point. In the second regulated step (the third step of glycolysis), phosphofructokinase converts fructose-6-phosphate into fructose-1,6-bisphosphate, which then is converted into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. The dihydroxyacetone phosphate can be removed from glycolysis by conversion into glycerol-3-phosphate, which can be used to form triglycerides.[10] On the converse, triglycerides can be broken down into fatty acids and glycerol; the latter, in turn, can be converted into dihydroxyacetone phosphate, which can enter glycolysis after the second control point.

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208

Regulation The three regulated enzymes are hexokinase, phosphofructokinase, and pyruvate kinase. The flux through the glycolytic pathway is adjusted in response to conditions both inside and outside the cell. The rate in liver is regulated to meet major cellular needs: (1) the production of ATP, (2) the provision of building blocks for biosynthetic reactions, and (3) to lower blood glucose, one of the major functions of the liver. When blood sugar falls, glycolysis is halted in the liver to allow the reverse process, gluconeogenesis. In glycolysis, the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase are effectively irreversible in most organisms. In metabolic pathways, such enzymes are potential sites of control, and all three enzymes serve this purpose in glycolysis. Hexokinase In animals, regulation of blood glucose levels by the pancreas in conjunction with the liver is a vital part of homeostasis. In liver cells, extra G6P (glucose-6-phosphate) may be converted to G1P for conversion to glycogen, or it is alternatively converted by glycolysis to acetyl-CoA and then citrate. Excess citrate is exported to the cytosol, where ATP citrate lyase will regenerate acetyl-CoA and OAA. The acetyl-CoA is then used for fatty acid synthesis and cholesterol synthesis, two important ways of utilizing excess glucose when its [11] Yeast hexokinase B. PDB 1IG8 . concentration is high in blood. Liver contains both hexokinase and glucokinase; both catalyse the phosphorylation of glucose to G6P but the latter is not inhibited by G6P. Thus, glucokinase allows glucose to be converted into glycogen, fatty acids, and cholesterol even as G6P accumulates in hepatocytes.[12] This is important when blood glucose levels are high. During hypoglycemia, the glycogen can be converted back to G6P and then converted to glucose by the liver-specific enzyme glucose 6-phosphatase and released into the blood without taking up the low concentration of glucose it releases. This reverse reaction is an important role of liver cells to maintain blood sugars levels during fasting. This is critical for brain function, since the brain utilizes glucose as an energy source under most conditions. Phosphofructokinase Phosphofructokinase is an important control point in the glycolytic pathway, since it is one of the irreversible steps and has key allosteric effectors, AMP and fructose 2,6-bisphosphate (F2,6BP). Fructose 2,6-bisphosphate (F2,6BP) is a very potent activator of phosphofructokinase (PFK-1), which is synthesised when F6P is phosphorylated by a second phosphofructokinase (PFK2). In liver, when blood sugar is low and glucagon elevates cAMP, PFK2 is phosphorylated by protein kinase A. The phosphorylation inactivates PFK2, and another domain on this protein becomes active as fructose 2,6-bisphosphatase, which converts F2,6BP back to F6P. Both Bacillus stearothermophilus phosphofructokinase. [13] glucagon and epinephrine cause high levels of cAMP in the liver. The PDB 6PFK . result of lower levels of liver fructose-2,6-bisphosphate is a decrease in activity of phosphofructokinase and an increase in activity of fructose 1,6-bisphosphatase, so that gluconeogenesis (in essence, "glycolysis in reverse") is favored. This is consistent with the role of the liver in such situations, since the response of the liver to these hormones is to release glucose to the blood.

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209

ATP competes with AMP for the allosteric effector site on the PFK enzyme. ATP concentrations in cells are much higher than those of AMP, typically 100-fold higher,[14] but the concentration of ATP does not change more than about 10% under physiological conditions, whereas a 10% drop in ATP results in a 6-fold increase in AMP.[15] Thus, the relevance of ATP as an allosteric effector is questionable. An increase in AMP is a consequence of a decrease in energy charge in the cell. Citrate inhibits phosphofructokinase when tested in vitro by enhancing the inhibitory effect of ATP. However, it is doubtful that this is a meaningful effect in vivo, because citrate in the cytosol is utilized mainly for conversion to acetyl-CoA for fatty acid and cholesterol synthesis. Pyruvate kinase This enzyme catalyzes the last step of glycolysis, in which pyruvate and ATP are formed. Regulation of this enzyme is discussed in the main topic, pyruvate kinase.

Post-glycolysis processes The overall process of glycolysis is: glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O If glycolysis were to continue indefinitely, all of the NAD+ would be used up, and glycolysis would stop. To allow glycolysis to continue, organisms must be able to oxidize NADH back to NAD+.

Yeast pyruvate kinase. PDB 1A3W

[16]

Fermentation One method of doing this is to simply have the pyruvate do the oxidation; in this process, the pyruvate is converted to lactate (the conjugate base of lactic acid) in a process called lactic acid fermentation: pyruvate + NADH + H+ → lactate + NAD+ This process occurs in the bacteria involved in making yogurt (the lactic acid causes the milk to curdle). This process also occurs in animals under hypoxic (or partially anaerobic) conditions, found, for example, in overworked muscles that are starved of oxygen, or in infarcted heart muscle cells. In many tissues, this is a cellular last resort for energy; most animal tissue cannot tolerate anaerobic conditions for an extended period of time. Some organisms, such as yeast, convert NADH back to NAD+ in a process called ethanol fermentation. In this process, the pyruvate is converted first to acetaldehyde and carbon dioxide, then to ethanol. Lactic acid fermentation and ethanol fermentation can occur in the absence of oxygen. This anaerobic fermentation allows many single-cell organisms to use glycolysis as their only energy source.

Anaerobic respiration In the above two examples of fermentation, NADH is oxidized by transferring two electrons to pyruvate. However, anaerobic bacteria use a wide variety of compounds as the terminal electron acceptors in cellular respiration: nitrogenous compounds, such as nitrates and nitrites; sulfur compounds, such as sulfates, sulfites, sulfur dioxide, and elemental sulfur; carbon dioxide; iron compounds; manganese compounds; cobalt compounds; and uranium compounds.

Glycolysis

Aerobic respiration In aerobic organisms, a complex mechanism has been developed to use the oxygen in air as the final electron acceptor. • First, pyruvate is converted to acetyl-CoA and CO2 within the mitochondria in a process called pyruvate decarboxylation. • Second, the acetyl-CoA enters the citric acid cycle, also known as Krebs Cycle, where it is fully oxidized to carbon dioxide and water, producing yet more NADH. • Third, the NADH is oxidized to NAD+ by the electron transport chain, using oxygen as the final electron acceptor. This process creates a hydrogen ion gradient across the inner membrane of the mitochondria. • Fourth, the proton gradient is used to produce about 2.5 ATP for every NADH oxidized in a process called oxidative phosphorylation.

Intermediates for other pathways This article concentrates on the catabolic role of glycolysis with regard to converting potential chemical energy to usable chemical energy during the oxidation of glucose to pyruvate. Many of the metabolites in the glycolytic pathway are also used by anabolic pathways, and, as a consequence, flux through the pathway is critical to maintain a supply of carbon skeletons for biosynthesis. In addition, not all carbon entering the pathway leaves as pyruvate and may be extracted at earlier stages to provide carbon compounds for other pathways. These metabolic pathways are all strongly reliant on glycolysis as a source of metabolites: and many more. • • • •

Gluconeogenesis Lipid metabolism Pentose phosphate pathway Citric acid cycle, which in turn leads to: • Amino acid synthesis • Nucleotide synthesis • Tetrapyrrole synthesis

From an anabolic metabolism perspective, the NADH has a role to drive synthetic reactions, doing so by directly or indirectly reducing the pool of NADP+ in the cell to NADPH, which is another important reducing agent for biosynthetic pathways in a cell.

Glycolysis in disease Genetic diseases Glycolytic mutations are generally rare due to importance of the metabolic pathway, this means that the majority of occurring mutations result in an inability for the cell to respire, and therefore cause the death of the cell at an early stage. However, some mutations are seen with one notable example being Pyruvate kinase deficiency, leading to chronic hemolytic anemia.

Cancer Malignant rapidly growing tumor cells typically have glycolytic rates that are up to 200 times higher than those of their normal tissues of origin. This phenomenon was first described in 1930 by Otto Warburg and is referred to as the Warburg effect. The Warburg hypothesis claims that cancer is primarily caused by dysfunctionality in mitochondrial metabolism, rather than because of uncontrolled growth of cells. A number of theories have been advanced to explain the Warburg effect. One such theory suggests that the increased glycolysis is a normal protective process of

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Glycolysis the body and that malignant change could be primarily caused by energy metabolism.[17] This high glycolysis rate has important medical applications, as high aerobic glycolysis by malignant tumors is utilized clinically to diagnose and monitor treatment responses of cancers by imaging uptake of 2-18F-2-deoxyglucose (FDG) (a radioactive modified hexokinase substrate) with positron emission tomography (PET).[18][19] There is ongoing research to affect mitochondrial metabolism and treat cancer by reducing glycolysis and thus starving cancerous cells in various new ways, including a ketogenic diet.

Alzheimer's disease Disfunctioning glycolysis or glucose metabolism in fronto-temporo-parietal and cingulate cortices has been associated with Alzheimer's disease,[] probably due to the decreased amyloid β (1-42) (Aβ42) and increased tau, phosphorylated tau in cerebrospinal fluid (CSF)[]

Interactive pathway map Click on genes, proteins and metabolites below to link to respective articles. [20]

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Glycolysis

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Glycolysis

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Glycolysis and Gluconeogenesis edit [21] [1] Webster's New International Dictionary of the English Language, 2nd ed. (1937) Merriam Company, Springfield, Mass. [3] Romano AH, Conway T. (1996) Evolution of carbohydrate metabolic pathways. Res Microbiol. 147(6–7):448–55 PMID 9084754 [4] Kim BH, Gadd GM. (2011) Bacterial Physiology and Metabolism, 3rd edition. [5] Glycolysis – Animation and Notes (http:/ / pharmaxchange. info/ press/ 2011/ 09/ glycolysis-animation-and-notes/ ) [11] http:/ / www. rcsb. org/ pdb/ explore/ explore. do?structureId=1IG8 [12] Voet D., and Voet J. G. (2004). Biochemistry 3rd Edition (New York, John Wiley & Sons, Inc.) [13] http:/ / www. rcsb. org/ pdb/ explore/ explore. do?structureId=6PFK [14] Beis I., and Newsholme E. A. (1975). The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates. Biochem J 152, 23-32. [15] Voet D., and Voet J. G. (2004). Biochemistry 3rd Edition (New York, John Wiley & Sons, Inc.). [16] http:/ / www. rcsb. org/ pdb/ explore/ explore. do?structureId=1A3W [20] The interactive pathway map can be edited at WikiPathways: [21] http:/ / www. wikipathways. org/ index. php/ Pathway:WP534

Alternative nomenclature Some of the metabolites in glycolysis have alternative names and nomenclature. In part, this is because some of them are common to other pathways, such as the Calvin cycle. This article

Alternative names

Alternative nomenclature

glucose

Glc

fructose 6-phosphate

F6P

fructose 1,6-bisphosphate

F1,6BP fructose 1,6-diphosphate

dihydroxyacetone phosphate DHAP

glycerone phosphate

glyceraldehyde 3-phosphate GADP

3-phosphoglyceraldehyde

1,3-bisphosphoglycerate

1,3BPG glycerate PGAP, BPG, DPG 1,3-bisphosphate, glycerate 1,3-diphosphate, 1,3-diphosphoglycerate

3-phosphoglycerate

3PG

glycerate 3-phosphate

2-phosphoglycerate

2PG

glycerate 2-phosphate

10 phosphoenolpyruvate

PEP

11 pyruvate

Pyr

dextrose

FBP, FDP, F1,6DP

PGAL, G3P, GALP,GAP,TP

PGA, GP

pyruvic acid

References External links • A Detailed Glycolysis Animation provided by [[IUBMB (http://www.iubmb-nicholson.org/swf/glycolysis. swf)]] ( Adobe Flash (http://get.adobe.com/flashplayer/) Required) • The Glycolytic enzymes in Glycolysis (http://nist.rcsb.org/pdb/molecules/pdb50_1.html) at RCSB PDB • Glycolytic cycle with animations (http://www.wdv.com/CellWorld/Biochemistry/Glycolytic) at wdv.com • Metabolism, Cellular Respiration and Photosynthesis - The Virtual Library of Biochemistry and Cell Biology (http://www.biochemweb.org/metabolism.shtml) at biochemweb.org • notes on glycolysis (http://www.rahulgladwin.com/blog/2007/01/notes-on-glycolysis.html) at rahulgladwin.com • The chemical logic behind glycolysis (http://www2.ufp.pt/~pedros/bq/glycolysis.htm) at ufp.pt

Glycolysis • Expasy biochemical pathways poster (http://www.expasy.org/tools/pathways/boehringer_legends.html) at ExPASy • Mnemonic at medicalmnemonics.com 317 5468 (http://www.medicalmnemonics.com/cgi-bin/lookup. cfm?id1=317&id2=5468&id3=&id4=)

Gluconeogenesis Gluconeogenesis (abbreviated GNG) is a metabolic pathway that results in the generation of glucose from non-carbohydrate carbon substrates such as pyruvate, lactate, glycerol, glucogenic amino acids, and odd-chain fatty acid. It is one of the two main mechanisms humans and many other animals use to keep blood glucose levels from dropping too low (hypoglycemia). The other means of maintaining blood glucose levels is through the degradation of glycogen (glycogenolysis).[1] Gluconeogenesis is a ubiquitous process, present in plants, animals, fungi, bacteria, and other microorganisms.[2] In vertebrates, gluconeogenesis takes place mainly in the liver and, to a lesser extent, in the cortex of kidneys. In ruminants, this tends to be a continuous process.[3] In many other animals, the process occurs during periods of fasting, starvation, low-carbohydrate diets, or intense exercise. The process is highly endergonic until ATP or GTP are utilized, effectively making the process exergonic. For example, the pathway leading from pyruvate to glucose-6-phosphate requires 4 molecules of ATP and 2 molecules of GTP. Gluconeogenesis is often associated with ketosis. Gluconeogenesis is also a target of therapy for type II diabetes, such as metformin, which inhibits glucose formation and stimulates glucose uptake by cells.[4] In ruminants, because metabolizable dietary carbohydrates tend to be metabolized by rumen organisms, gluconeogenesis occurs regardless of fasting, low-carbohydrate diets, exercise, etc.[5]

Precursors In humans the main gluconeogenic precursors are lactate, glycerol (which is a part of the triacylglycerol molecule), alanine and glutamine. Altogether, they account for over 90% of the overall gluconeogenesis.[] Other glucogenic amino acid as well as all citric acid cycle intermediates, the latter through conversion to oxaloacetate, can also function as substrates for gluconeogenesis.[] In ruminants, propionate is the principal gluconeogenic substrate.[5][7] Lactate is transported back to the liver Catabolism of proteinogenic amino acids. Amino acids are classified according the where it is converted into pyruvate by the [6] abilities of their products to enter gluconeogenesis: Glucogenic amino acids have Cori cycle using the enzyme lactate this abilityKetogenic amino acids do not. These products may still be used for dehydrogenase. Pyruvate, the first ketogenesis or lipid synthesis.Some amino acids are catabolized into both designated substrate of the gluconeogenic glucogenic and ketogenic products. pathway, can then be used to generate glucose.[] Transamination or deamination of amino acids facilitates entering of their carbon skeleton into the cycle directly (as pyruvate or oxaloacetate), or indirectly via the citric acid cycle.

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Gluconeogenesis Whether even-chain fatty acids can be converted into glucose in animals has been a longstanding question in biochemistry.[] It is known that odd-chain fatty acids can be oxidized to yield propionyl CoA, a precursor for succinyl CoA, which can be converted to pyruvate and enter into gluconeogenesis. In plants, specifically seedlings, the glyoxylate cycle can be used to convert fatty acids (acetate) into the primary carbon source of the organism. The glyoxylate cycle produces four-carbon dicarboxylic acids that can enter gluconeogenesis.[] In 1995, researchers identified the glyoxylate cycle in nematodes.[] In addition, the glyoxylate enzymes malate synthase and isocitrate lyase have been found in animal tissues.[] Genes coding for malate synthase gene have been identified in other [metazoans] including arthropods, echinoderms, and even some vertebrates. Mammals found to possess these genes include monotremes (platypus) and marsupials (opossum) but not placental mammals. Genes for isocitrate lyase are found only in nematodes, in which, it is apparent, they originated in horizontal gene transfer from bacteria. The existence of glyoxylate cycles in humans has not been established, and it is widely held that fatty acids cannot be converted to glucose in humans directly. However, carbon-14 has been shown to end up in glucose when it is supplied in fatty acids.[] Despite these findings, it is considered unlikely that the 2-carbon acetyl-CoA derived from the oxidation of fatty acids would produce a net yield of glucose via the citric acid cycle.[]

Location In mammals, gluconeogenesis is restricted to the liver,[] the kidney[] and the intestine.[] However these organs use somewhat different gluconeogenic precursors. Liver uses primarily lactate and alanine while kidney uses lactate and glutamine.[] Propionate is the principal substrate for gluconeogenesis in the ruminant liver, and the ruminant liver may make increased use of gluconeogenic amino acids, e.g. alanine, when glucose demand is increased.[8] The capacity of liver cells to use lactate for gluconeogenesis declines from the preruminant stage to the ruminant stage in calves and lambs.[9] In sheep kidney tissue, very high rates of gluconeogenesis from propionate have been observed.[10] The intestine uses mostly glutamine and glycerol.[] In all species, the formation of oxaloacetate from pyruvate and TCA cycle intermediates is restricted to the mitochondrion, and the enzymes that convert Phosphoenolpyruvic acid (PEP) to glucose are found in the cytosol.[] The location of the enzyme that links these two parts of gluconeogenesis by converting oxaloacetate to PEP, PEP carboxykinase, is variable by species: it can be found entirely within the mitochondria, entirely within the cytosol, or dispersed evenly between the two, as it is in humans.[] Transport of PEP across the mitochondrial membrane is accomplished by dedicated transport proteins; however no such proteins exist for oxaloacetate.[] Therefore, in species that lack intra-mitochondrial PEP, oxaloacetate must be converted into malate or asparate, exported from the mitochondrion, and converted back into oxaloacetate in order to allow gluconeogenesis to continue.[]

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Gluconeogenesis

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Pathway Gluconeogenesis is a pathway consisting of a series of eleven enzyme-catalyzed reactions. The pathway may begin in the mitochondria or cytoplasm, this being dependent on the substrate being used. Many of the reactions are the reversible steps found in glycolysis. • Gluconeogenesis begins in the mitochondria with the formation of oxaloacetate by the carboxylation of pyruvate. This reaction also requires one molecule of ATP, and is catalyzed by pyruvate carboxylase. This enzyme is stimulated by high levels of acetyl-CoA (produced in β-oxidation in the liver) and inhibited by high levels of ADP. • Oxaloacetate is reduced to malate using NADH, a step required for its transportation out of the mitochondria. • Malate is oxidized to oxaloacetate using NAD+ in the cytosol, where the remaining steps of gluconeogenesis take place. • Oxaloacetate is decarboxylated and then phosphorylated to form phosphoenolpyruvate using the enzyme phosphoenolpyruvate carboxykinase. A molecule of GTP is hydrolyzed to GDP during this reaction. • The next steps in the reaction are the same as reversed glycolysis. However, fructose-1,6-bisphosphatase converts fructose-1,6-bisphosphate to fructose 6-phosphate, using one water molecule and releasing one phosphate. This is also the rate-limiting step of gluconeogenesis.

Gluconeogenesis pathway with key molecules and enzymes. Many steps are the opposite of those found in the glycolysis.

• Glucose-6-phosphate is formed from fructose 6-phosphate by phosphoglucoisomerase. Glucose-6-phosphate can be used in other metabolic pathways or dephosphorylated to free glucose. Whereas free glucose can easily diffuse in and out of the cell, the phosphorylated form (glucose-6-phosphate) is locked in the cell, a mechanism by which intracellular glucose levels are controlled by cells. • The final reaction of gluconeogenesis, the formation of glucose, occurs in the lumen of the endoplasmic reticulum, where glucose-6-phosphate is hydrolyzed by glucose-6-phosphatase to produce glucose. Glucose is shuttled into the cytoplasm by glucose transporters located in the endoplasmic reticulum's membrane. Metabolism of common monosaccharides, including glycolysis, gluconeogenesis, glycogenesis and glycogenolysis

Gluconeogenesis

Regulation While most steps in gluconeogenesis are the reverse of those found in glycolysis, three regulated and strongly exergonic reactions are replaced with more kinetically favorable reactions. Hexokinase/glucokinase, phosphofructokinase, and pyruvate kinase enzymes of glycolysis are replaced with glucose-6-phosphatase, fructose-1,6-bisphosphatase, and PEP carboxykinase. This system of reciprocal control allow glycolysis and gluconeogenesis to inhibit each other and prevent the formation of a futile cycle. The majority of the enzymes responsible for gluconeogenesis are found in the cytoplasm; the exceptions are mitochondrial pyruvate carboxylase and, in animals, phosphoenolpyruvate carboxykinase. The latter exists as an isozyme located in both the mitochondrion and the cytosol.[11] The rate of gluconeogenesis is ultimately controlled by the action of a key enzyme, fructose-1,6-bisphosphatase, which is also regulated through signal transduction by cAMP and its phosphorylation. Most factors that regulate the activity of the gluconeogenesis pathway do so by inhibiting the activity or expression of key enzymes. However, both acetyl CoA and citrate activate gluconeogenesis enzymes (pyruvate carboxylase and fructose-1,6-bisphosphatase, respectively). Due to the reciprocal control of the cycle, acetyl-CoA and citrate also have inhibitory roles in the activity of pyruvate kinase. Global control of gluconeogenesis is mediated by glucagon (released when blood glucose is low); it triggers phosphorylation of enzymes and regulatory proteins by Protein Kinase A (a cyclic AMP regulated kinase) resulting in inhibition of glycolysis and stimulation of gluconeogenesis. Recent studies have shown that the absence of hepatic glucose production has no major effect on the control of fasting plasma glucose concentration. Compensatory induction of gluconeogenesis occurs in the kidneys and intestine, driven by glucagon, glucocorticoids, and acidosis.[]

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References [3] Young, J. W. 1977. Gluconeogenesis in cattle: significance and methodology. J. Dairy Sci. 60: 1-15. [5] Beitz, D. C. 2004. Carbohydrate metabolism. In: Reese, W. O. Dukes' physiology of domestic animals. 12th ed. Cornell Univ. Press. pp. 501-515. [6] Chapter 20 (Amino Acid Degradation and Synthesis) in: [7] Van Soest, P. J. 1994. Nutritional ecology of the ruminant. 2nd Ed. Cornell Univ. Press. 476 pp. [8] Overton, T. R., J. K. Drackley, C. J. Ottemann-Abbamonte, A. D. Beaulieu, L. S. Emmert and J. H. Clark. 1999. Substrate utilization for hepatic gluconeogenesis is altered by increased glucose demand in ruminants. J. Anim. Sci. 77: 1940-1951. [9] Donkin, S. S. and L. E. Armentano. 1995. Insulin and glucagon regulation of gluconeogenesis in preruminating and ruminating bovine. J. Anim. Sci. 73: 546-551. [10] Sasaki, S., K. Ambo, M. Muramatsu and T. Tsuda. 1975. Gluconeogenesis in the kidney-cortex slices of normal fed and starved sheep. Tohoku J. Agr. Res. 26: 20-29. [11] Chakravarty, K., Cassuto, H., Resef, L., & Hanson, R.W. (2005) Factors that control the tissue-specific transcription of the gene for phosphoenolpyruvate carboxykinase-C. Critical Reviews of Biochemistry and Molecular Biology, 40(3), 129-154.

External links • Overview at indstate.edu (http://themedicalbiochemistrypage.org/gluconeogenesis.html) • Interactive diagram at uakron.edu (http://ull.chemistry.uakron.edu/Pathways/gluconeogenesis/index.html#) • The chemical logic behind gluconeogenesis (http://homepage.ufp.pt/pedros/bq/gng.htm)

Glycogen Glycogen is a multibranched polysaccharide that serves as a form of energy storage in animals[2] and fungi. In humans, glycogen is made and stored primarily in the cells of the liver and the muscles, and functions as the secondary long-term energy storage (with the primary energy stores being fats held in adipose tissue). Glycogen is the analogue of starch, a glucose polymer in plants, and is sometimes referred to as animal starch, having a similar structure to amylopectin but more extensively branched and compact than starch. Glycogen is found in the form of granules in the cytosol/cytoplasm in many cell types, and plays an important role in the glucose cycle. Glycogen forms an energy reserve that can be quickly mobilized to meet a sudden need for glucose, but one that is less compact than the energy reserves of triglycerides (lipids).

Schematic 2-D cross-sectional view of glycogen. A core protein of glycogenin is surrounded by branches of glucose units. The entire [1] globular granule may contain approximately 30,000 glucose units.

Polysaccharide represents the main storage form of glucose in the body. Found in the liver and muscles, muscle glycogen is converted into glucose by muscle cells, and liver glycogen converts to glucose for use throughout the body including the Central Nervous System.

Glycogen

219

In the liver cells (hepatocyte)s, glycogen can compose up to eight percent of the fresh weight (100–120g in an adult) soon after a meal.[3] Only the glycogen stored in the liver can be made accessible to other organs. In the muscles, glycogen is found in a low concentration (one to two percent of the muscle mass). The amount of glycogen stored in the body—especially within the muscles, liver, and red blood cells[4][5][6]—mostly depends on physical training, basal metabolic rate, and eating habits such as intermittent fasting. Small amounts of glycogen are found in the kidneys, and even smaller amounts in certain glial cells in the brain and white blood cells. The uterus also stores glycogen during pregnancy to nourish the embryo.[7] A view of the atomic structure of a single branched strand of glucose units in a glycogen molecule.

Structure Glycogen is a branched biopolymer consisting of linear chains of glucose residues with further chains branching off every ten glucoses or so. Glucoses are linked together linearly by α(1→4) glycosidic bonds from one glucose to the next. Branches are linked to the chains they are branching off from by α(1→6) glycosidic bonds between the first glucose of the new branch and a glucose on the stem chain.[8]

Schematic of glycogen structure

Due to the way that glycogen is synthesised, every glycogen granule has at its core a glycogenin protein.[9]

Function Liver As a meal containing carbohydrates is eaten and digested, blood glucose levels rise, and the pancreas secretes insulin. Blood glucose from the portal vein enters liver cells (hepatocytes). Insulin acts on the hepatocytes to stimulate the action of several enzymes, including glycogen synthase. Glucose molecules are added to the chains of glycogen as long as both insulin and glucose remain plentiful. In this postprandial or "fed" state, the liver takes in more glucose from the blood than it releases. After a meal has been digested and glucose levels begin to fall, insulin secretion is reduced, and glycogen synthesis stops. When it is needed for energy, glycogen is broken down and converted again to glucose. Glycogen phosphorylase is the primary enzyme of glycogen breakdown. For the next 8–12 hours, glucose derived from liver

Glycogen glycogen will be the primary source of blood glucose to be used by the rest of the body for fuel. Glucagon is another hormone produced by the pancreas, which in many respects serves as a counter-signal to insulin. In response to insulin level below normal (when blood levels of glucose begin to fall below the normal range), glucagon is secreted in increasing amounts to stimulate glycogenolysis and gluconeogenesis pathways.

Muscle Muscle cell glycogen appears to function as an immediate reserve source of available glucose for muscle cells. Other cells that contain small amounts use it locally as well. Muscle cells lack the enzyme glucose-6-phosphatase, which is required to pass glucose into the blood, so the glycogen they store is destined for internal use and is not shared with other cells. (This is in contrast to liver cells, which, on demand, readily do break down their stored glycogen into glucose and send it through the blood stream as fuel for the brain or muscles). Glycogen is also a suitable storage substance due to its insolubility in water, which means it does not affect the osmotistic levels and pressure of a cell.

History Glycogen was discovered by Claude Bernard. His experiments showed that the liver contained a substance that could give rise to reducing sugar by the action of a "ferment" in the liver. By 1857 he described the isolation of a substance that he called "la matière glycogène", or "sugar-forming substance". Soon after the discovery of glycogen in the liver, A. Sanson found that muscular tissue also contains glycogen. The empirical formula for glycogen of (C6H10O5)n was established by Kekule in 1858.[10]

Metabolism Synthesis Glycogen synthesis is, unlike its breakdown, endergonic. This means that glycogen synthesis requires the input of energy. Energy for glycogen synthesis comes from UTP, which reacts with glucose-1-phosphate, forming UDP-glucose, in a reaction catalysed by UDP-glucose pyrophosphorylase. Glycogen is synthesized from monomers of UDP-glucose by the enzyme glycogen synthase, which progressively lengthens the glycogen chain with (α1→4) bonded glucose. As glycogen synthase can lengthen only an existing chain, the protein glycogenin is needed to initiate the synthesis of glycogen. The glycogen-branching enzyme, amylo (α1→4) to (α1→6) transglycosylase, catalyzes the transfer of a terminal fragment of 6-7 glucose residues from a nonreducing end to the C-6 hydroxyl group of a glucose residue deeper into the interior of the glycogen molecule. The branching enzyme can act upon only a branch having at least 11 residues, and the enzyme may transfer to the same glucose chain or adjacent glucose chains.

Breakdown Glycogen is cleaved from the nonreducing ends of the chain by the enzyme glycogen phosphorylase to produce monomers of glucose-1-phosphate, which is then converted to glucose 6-phosphate Action of Glycogen Phosphorylase on Glycogen by phosphoglucomutase. A special debranching enzyme is needed to remove the alpha(1-6) branches in branched glycogen and reshape the chain into linear polymer. The G6P monomers produced have three possible fates: • G6P can continue on the glycolysis pathway and be used as fuel. • G6P can enter the pentose phosphate pathway via the enzyme glucose-6-phosphate dehydrogenase to produce NADPH and 5-carbon sugars.

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Glycogen • In the liver and kidney, G6P can be dephosphorylated back to glucose by the enzyme glucose 6-phosphatase. This is the final step in the gluconeogenesis pathway.

Clinical relevance Disorders of glycogen metabolism The most common disease in which glycogen metabolism becomes abnormal is diabetes, in which, because of abnormal amounts of insulin, liver glycogen can be abnormally accumulated or depleted. Restoration of normal glucose metabolism usually normalizes glycogen metabolism as well. In hypoglycemia caused by excessive insulin, liver glycogen levels are high, but the high insulin level prevents the glycogenolysis necessary to maintain normal blood sugar levels. Glucagon is a common treatment for this type of hypoglycemia. Various inborn errors of metabolism are caused by deficiencies of enzymes necessary for glycogen synthesis or breakdown. These are collectively referred to as glycogen storage diseases.

Glycogen depletion and endurance exercise Long-distance athletes such as marathon runners, cross-country skiers, and cyclists often experience glycogen depletion, where almost all of the athlete's glycogen stores are depleted after long periods of exertion without enough energy consumption. This phenomenon is referred to as "hitting the wall". Glycogen depletion can be forestalled in three possible ways. First, during exercise carbohydrates with the highest possible rate of conversion to blood glucose per time (high glycemic Index) are ingested continuously. The best possible outcome of this strategy replaces about 35% of glucose consumed at heart rates above about 80% of maximum. Second, through training, the body can be conditioned to burn fat earlier, faster, and more efficiently[citation needed], sparing carbohydrate use from all sources. Third, by consuming foods low on the glycemic Index for 12–18 hours before the event, the liver and muscles will store the resulting slow but steady stream of glucose as glycogen, instead of fat. This process is known as carbohydrate loading. When experiencing glycogen debt, athletes often experience extreme fatigue to the point that it is difficult to move. As a reference, the very best professional cyclists in the world will usually finish a 4-5hr stage race right at the limit of glycogen depletion using the first 3 strategies. A study published in the Journal of Applied Physiology (online May 8, 2008) suggests that, when athletes ingest both carbohydrate and caffeine following exhaustive exercise, their glycogen is replenished more rapidly.Wikipedia:Identifying reliable sources (medicine)[11][12]

References External links • Glycogen detection using Periodic Acid Schiff Staining (http://www.histochem.net/protocol periodic acid schiff.htm) • Glycogen storage disease - McArdle's Disease Website (http://mcardlesdisease.org) • Glycogen (http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=&term=Glycogen) at the US National Library of Medicine Medical Subject Headings (MeSH)

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Pentose phosphate pathway

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Pentose phosphate pathway The pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt) is a process that generates NADPH and pentoses (5-carbon sugars). There are two distinct phases in the pathway. The first is the oxidative phase, in which NADPH is generated, and the second is the non-oxidative synthesis of 5-carbon sugars. This pathway is an alternative to glycolysis. While it does involve oxidation of glucose, its primary role is anabolic rather than catabolic. For most organisms, it takes place in the cytosol; in plants, most steps take place in plastids.[1]

Outcome The primary results of the Pathway are: • The generation of reducing equivalents, in the form of NADPH, used in reductive biosynthesis reactions within cells. (e.g. fatty acid synthesis) • Production of ribose-5-phosphate (R5P), used in the synthesis of nucleotides and nucleic acids. • Production of erythrose-4-phosphate (E4P), used in the synthesis of aromatic amino acids. Aromatic amino acids, in turn, are precursors for many biosynthetic pathways, notably including the lignin in wood.

The Pentose Phosphate Pathway

Dietary pentose sugars derived from the digestion of nucleic acids may be metabolized through the pentose phosphate pathway, and the carbon skeletons of dietary carbohydrates may be converted into glycolytic/gluconeogenic intermediates. In mammals, the PPP occurs exclusively in the cytoplasm, and is found to be most active in the liver, mammary gland and adrenal cortex in the human. The PPP is one of the three main ways the body creates molecules with reducing power, accounting for approximately 60% of NADPH production in humans. One of the uses of NADPH in the cell is to prevent oxidative stress. It reduces glutathione via glutathione reductase, which converts reactive H2O2 into H2O by glutathione peroxidase. If absent, the H2O2 would be converted to hydroxyl free radicals by Fenton chemistry, which can attack the cell. Erythrocytes, for example, generate a large amount of NADPH through the pentose phosphate pathway to use in the reduction of glutathione. Hydrogen peroxide is also generated for phagocytes in a process often referred to as a respiratory burst.[2]

Pentose phosphate pathway

223

Phases Oxidative phase In this phase, two molecules of NADP+ are reduced to NADPH, utilizing the energy from the conversion of glucose-6-phosphate into ribulose 5-phosphate.

Oxidative phase of pentose phosphate pathway. glucose-6-phosphate (1), 6-phosphoglucono-δ-lactone (2), 6-phosphogluconate (3), ribulose 5-phosphate (4).

The entire set of reactions can be summarized as follows: Reactants

Products

Enzyme

Description

Glucose 6-phosphate + NADP+

→ 6-phosphoglucono-δ-lactone + NADPH

glucose 6-phosphate dehydrogenase

Dehydrogenation. The hemiacetal hydroxyl group located on carbon 1 of glucose 6-phosphate is converted into a carbonyl group, generating a lactone, and, in the process, NADPH is generated.

6-phosphoglucono-δ-lactone + H2O

→ 6-phosphogluconate + H+

6-phosphogluconolactonase

Hydrolysis

6-phosphogluconate + NADP+

→ ribulose 5-phosphate + NADPH + CO2

6-phosphogluconate dehydrogenase

Oxidative decarboxylation. NADP+ is the electron acceptor, generating another molecule of NADPH, a CO2, and ribulose 5-phosphate.

The overall reaction for this process is: Glucose 6-phosphate + 2 NADP+ + H2O → ribulose 5-phosphate + 2 NADPH + 2 H+ + CO2

Pentose phosphate pathway

224

Non-oxidative phase

The pentose phosphate pathway's nonoxidative phase

Reactants

Products

Enzymes

ribulose 5-phosphate

→ ribose 5-phosphate

Ribulose 5-Phosphate Isomerase

ribulose 5-phosphate

→ xylulose 5-phosphate

Ribulose 5-Phosphate 3-Epimerase

xylulose 5-phosphate + ribose 5-phosphate

→ glyceraldehyde 3-phosphate + sedoheptulose 7-phosphate

transketolase

sedoheptulose 7-phosphate + glyceraldehyde 3-phosphate

→ erythrose 4-phosphate + fructose 6-phosphate

transaldolase

xylulose 5-phosphate + erythrose 4-phosphate

→ glyceraldehyde 3-phosphate + fructose 6-phosphate

transketolase

Net reaction: 3 ribulose-5-phosphate → 1 ribose-5-phosphate + 2 xylulose-5-phosphate → 2 fructose-6-phosphate + glyceraldehyde-3-phosphate

Regulation Glucose-6-phosphate dehydrogenase is the rate-controlling enzyme of this pathway. It is allosterically stimulated by NADP+. The ratio of NADPH:NADP+ is normally about 100:1 in liver cytosol. This makes the cytosol a highly-reducing environment. An NADPH-utilizing pathway forms NADP+, which stimulates Glucose-6-phosphate dehydrogenase to produce more NADPH. This step is also inhibited by acetyl CoA.

Erythrocytes and the pentose phosphate pathway Several deficiencies in the level of activity (not function) of glucose-6-phosphate dehydrogenase have been observed to be associated with resistance to the malarial parasite Plasmodium falciparum among individuals of Mediterranean and African descent. The basis for this resistance may be a weakening of the red cell membrane (the erythrocyte is the host cell for the parasite) such that it cannot sustain the parasitic life cycle long enough for productive growth.[3]

Pentose phosphate pathway

References External links • The chemical logic behind the pentose phosphate pathway (http://www2.ufp.pt/~pedros/bq/ppp.htm) • Pentose Phosphate Pathway (http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=&term=Pentose+ Phosphate+Pathway) at the US National Library of Medicine Medical Subject Headings (MeSH) • Pentose phosphate pathway Map - Homo sapiens (http://www.genome.jp/dbget-bin/ www_bget?path:hsa00030)

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Citric acid cycle Citric acid cycle The citric acid cycle — also known as the tricarboxylic acid cycle (TCA cycle), or the Krebs cycle.[][] — is a series of chemical reactions used by all aerobic organisms to generate energy through the oxidization of acetate derived from carbohydrates, fats and proteins into carbon dioxide. In addition, the cycle provides precursors including certain amino acids as well as the reducing agent NADH that is used in numerous biochemical reactions. Its central importance to many biochemical pathways suggests that it was one of the earliest established components of cellular metabolism and may have originated abiogenically.[]

Overview of the citric acid cycle (click to enlarge)

The name of this metabolic pathway is derived from citric acid (a type of tricarboxylic acid) that is first consumed and then regenerated by this sequence of reactions to complete the cycle. In addition, the cycle consumes acetate (in the form of acetyl-CoA) and water, reduces NAD+ to NADH, and produces carbon dioxide. The NADH generated by the TCA cycle is fed into the oxidative phosphorylation pathway. The net result of these two closely linked pathways is the oxidation of nutrients to produce usable energy in the form of ATP. In eukaryotic cells, the citric acid cycle occurs in the matrix of the mitochondrion. Bacteria also use the TCA cycle to generate energy, but since they lack mitochondria, the reaction sequence is performed in the cytosol with the proton gradient for ATP production being across the plasma membrane rather than the inner membrane of the mitochondrion. Several of the components and reactions of the citric acid cycle were established in the 1930s by the research of the Nobel laureate Albert Szent-Györgyi, for which he received the Nobel Prize in 1937 for his discoveries pertaining to fumaric acid, a key component of the cycle.[] The citric acid cycle itself was finally identified in 1937 by Hans Adolf Krebs whilst at the University of Sheffield, for which he received the Nobel Prize for Physiology or Medicine in 1953.[]

Citric acid cycle

Evolution Components of the TCA cycle were derived from anaerobic bacteria, and the TCA cycle itself may have evolved more than once.[] Theoretically there are several alternatives to the TCA cycle, however the TCA cycle appears to be the most efficient. If several TCA alternatives had independently evolved, they all appear to have converged onto the canonical TCA cycle.[][]

Overview The citric acid cycle is a key component of the metabolic pathway by which all aerobic organisms generate energy. Through catabolism of sugars, fats, and proteins, a two carbon organic product acetate in the form of acetyl-CoA is produced. Acetyl-CoA along with two equivalents of water (H2O) is consumed by the citric acid cycle producing two equivalents of carbon dioxide (CO2) and one equivalent of HS-CoA. In addition, one complete turn of the cycle converts three equivalents of nicotinamide adenine dinucleotide (NAD+) into three equivalents of reduced NAD+ (NADH), one equivalent of ubiquinone (Q) into one equivalent of reduced ubiquinone (QH2), and one equivalent each of guanosine diphosphate (GDP) and inorganic phosphate (Pi) into one equivalent of guanosine triphosphate (GTP). The NADH and QH2 generated by the citric acid cycle are in turn used by the oxidative phosphorylation pathway to generate energy-rich adenosine triphosphate (ATP). One of the primary sources of acetyl-CoA is sugars that are broken down by glycolysis to produce pyruvate that in turn is decarboxylated by the enzyme pyruvate dehydrogenase generating acetyl-CoA according to the following reaction scheme: • CH3C(=O)C(=O)O– (pyruvate) + HSCoA + NAD+ → CH3C(=O)SCoA (acetyl-CoA) + NADH + CO2 The product of this reaction, acetyl-CoA, is the starting point for the citric acid cycle. Below is a schematic outline of the cycle: • The citric acid cycle begins with the transfer of a two-carbon acetyl group from acetyl-CoA to the four-carbon acceptor compound (oxaloacetate) to form a six-carbon compound (citrate). • The citrate then goes through a series of chemical transformations, losing two carboxyl groups as CO2. The carbons lost as CO2 originate from what was oxaloacetate, not directly from acetyl-CoA. The carbons donated by acetyl-CoA become part of the oxaloacetate carbon backbone after the first turn of the citric acid cycle. Loss of the acetyl-CoA-donated carbons as CO2 requires several turns of the citric acid cycle. However, because of the role of the citric acid cycle in anabolism, they may not be lost, since many TCA cycle intermediates are also used as precursors for the biosynthesis of other molecules.[] • Most of the energy made available by the oxidative steps of the cycle is transferred as energy-rich electrons to NAD+, forming NADH. For each acetyl group that enters the citric acid cycle, three molecules of NADH are produced. • Electrons are also transferred to the electron acceptor Q, forming QH2. • At the end of each cycle, the four-carbon oxaloacetate has been regenerated, and the cycle continues.

Steps Two carbon atoms are oxidized to CO2, the energy from these reactions being transferred to other metabolic processes by GTP (or ATP), and as electrons in NADH and QH2. The NADH generated in the TCA cycle may later donate its electrons in oxidative phosphorylation to drive ATP synthesis; FADH2 is covalently attached to succinate dehydrogenase, an enzyme functioning both in the TCA cycle and the mitochondrial electron transport chain in oxidative phosphorylation. FADH2, therefore, facilitates transfer of electrons to coenzyme Q, which is the final electron acceptor of the reaction catalyzed by the Succinate:ubiquinone oxidoreductase complex, also acting as an intermediate in the electron transport chain.[] The citric acid cycle is continuously supplied with new carbon in the form of acetyl-CoA, entering at step 1 below.[]

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Citric acid cycle

Substrates

228

Products

Enzyme

Reaction type

Comment

Oxaloacetate + Acetyl CoA + H2O

Citrate + CoA-SH

Citrate synthase

Aldol condensation

irreversible, extends the 4C oxaloacetate to a 6C molecule

Citrate

cis-Aconitate + H2O

Aconitase

Dehydration

reversible isomerisation

cis-Aconitate + H2O

Isocitrate

Isocitrate + NAD+

Oxalosuccinate + NADH + H +

Oxalosuccinate

Hydration

Oxidation

generates NADH (equivalent of 2.5 ATP)

α-Ketoglutarate + CO2

Decarboxylation

rate-limiting, irreversible stage, generates a 5C molecule

α-Ketoglutarate Succinyl-CoA + α-Ketoglutarate dehydrogenase + NADH + H+ + + NAD + CO2 CoA-SH

Oxidative decarboxylation

irreversible stage, generates NADH (equivalent of 2.5 ATP), regenerates the 4C chain (CoA excluded)

Succinyl-CoA + Succinate + GDP + Pi CoA-SH + GTP

substrate-level phosphorylation

[] or ADP→ATP instead of GDP→GTP, generates 1 ATP or equivalent Condensation reaction of GDP + Pi and hydrolysis of Succinyl-CoA involve the H2O needed for balanced equation.

Succinate + ubiquinone (Q)

Fumarate + Succinate ubiquinol (QH2) dehydrogenase

Oxidation

uses FAD as a prosthetic group (FAD→FADH2 in the [] first step of the reaction) in the enzyme, generates the equivalent of 1.5 ATP

Fumarate + H2O

L-Malate

Fumarase

Hydration

Oxaloacetate + NADH + H+

Malate dehydrogenase

Oxidation

10 L-Malate + NAD+

Isocitrate dehydrogenase

Succinyl-CoA synthetase

reversible (in fact, equilibrium favors malate), generates NADH (equivalent of 2.5 ATP)

Mitochondria in animals, including humans, possess two succinyl-CoA synthetases: one that produces GTP from GDP, and another that produces ATP from ADP.[] Plants have the type that produces ATP (ADP-forming succinyl-CoA synthetase).[] Several of the enzymes in the cycle may be loosely associated in a multienzyme protein complex within the mitochondrial matrix.[] The GTP that is formed by GDP-forming succinyl-CoA synthetase may be utilized by nucleoside-diphosphate kinase to form ATP (the catalyzed reaction is GTP + ADP → GDP + ATP).[]

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Products Products of the first turn of the cycle are: one GTP (or ATP), three NADH, one QH2, two CO2. Because two acetyl-CoA molecules are produced from each glucose molecule, two cycles are required per glucose molecule. Therefore, at the end of two cycles, the products are: two GTP, six NADH, two QH2, and four CO2 Description

Reactants

Products

The sum of all reactions in the citric acid cycle is:

Acetyl-CoA + 3 NAD+ + Q + GDP + Pi + 3 H2O

→ CoA-SH + 3 NADH + 3 H+ + QH2 + GTP + 2 CO2

Combining the reactions occurring during the pyruvate oxidation with those occurring during the citric acid cycle, the following overall pyruvate oxidation reaction is obtained:

Pyruvate ion + 4 NAD+ + Q + GDP + Pi + 2 H2O

→ 4 NADH + 4 H+ + QH2 + GTP + 3 CO2

Combining the above reaction with the ones occurring in the course of glycolysis, the following overall glucose oxidation reaction (excluding reactions in the respiratory chain) is obtained:

Glucose + 10 NAD+ + 2 Q + 2 ADP + 2 GDP + 4 Pi + 2 H2O

→ 10 NADH + 10 H+ + 2 QH2 + 2 ATP + 2 GTP + 6 CO2

The above reactions are balanced if Pi represents the H2PO4- ion, ADP and GDP the ADP2- and GDP2- ions, respectively, and ATP and GTP the ATP3- and GTP3- ions, respectively. The total number of ATP obtained after complete oxidation of one glucose in glycolysis, citric acid cycle, and oxidative phosphorylation is estimated to be between 30 and 38.[]

Efficiency The theoretical maximum yield of ATP through oxidation of one molecule of glucose in glycolysis, citric acid cycle, and oxidative phosphorylation is 38 (assuming 3 molar equivalents of ATP per equivalent NADH and 2 ATP per FADH2). In eukaryotes, two equivalents of NADH are generated in glycolysis, which occurs in the cytoplasm. Transport of these two equivalents into the mitochondria consumes two equivalents of ATP, thus reducing the net production of ATP to 36. Furthermore, inefficiencies in oxidative phosphorylation due to leakage of protons across of the mitochondrial membrane and slippage of the ATP synthase/proton pump commonly reduces the ATP yield from NADH and FADH2 to less than the theoretical maximum yield.[] The observed yields are, therefore, closer to ~2.5 ATP per NADH and ~1.5 ATP per FADH2, further reducing the total net production of ATP to approximately 30.[] An assessment of the total ATP yield with newly revised proton-to-ATP ratios provides an estimate of 29.85 ATP per glucose molecule.[]

Regulation The regulation of the TCA cycle is largely determined by product inhibition and substrate availability. NADH, a product of all dehydrogenases in the TCA cycle with the exception of succinate dehydrogenase, inhibits pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and also citrate synthase. Acetyl-coA inhibits pyruvate dehydrogenase, while succinyl-CoA inhibits alpha-ketoglutarate dehydrogenase and citrate synthase. When tested in vitro with TCA enzymes, ATP inhibits citrate synthase and α-ketoglutarate dehydrogenase; however, ATP levels do not change more than 10% in vivo between rest and vigorous exercise. There is no known allosteric mechanism that can account for large changes in reaction rate from an allosteric effector whose concentration changes less than 10%.[1] Calcium is used as a regulator. It activates pyruvate dehydrogenase phosphatase which in turn activates the pyruvate dehydrogenase complex. Calcium also activates isocitrate dehydrogenase and α-ketoglutarate dehydrogenase.[] This increases the reaction rate of many of the steps in the cycle, and therefore increases flux throughout the pathway.

Citric acid cycle Citrate is used for feedback inhibition, as it inhibits phosphofructokinase, an enzyme involved in glycolysis that catalyses formation of fructose 1,6-bisphosphate,a precursor of pyruvate. This prevents a constant high rate of flux when there is an accumulation of citrate and a decrease in substrate for the enzyme. Recent work has demonstrated an important link between intermediates of the citric acid cycle and the regulation of hypoxia-inducible factors (HIF). HIF plays a role in the regulation of oxygen homeostasis, and is a transcription factor that targets angiogenesis, vascular remodeling, glucose utilization, iron transport and apoptosis. HIF is synthesized consititutively, and hydroxylation of at least one of two critical proline residues mediates their interaction with the von Hippel Lindau E3 ubiquitin ligase complex, which targets them for rapid degradation. This reaction is catalysed by prolyl 4-hydroxylases. Fumarate and succinate have been identified as potent inhibitors of prolyl hydroxylases, thus leading to the stabilisation of HIF.[]

Major metabolic pathways converging on the TCA cycle Several catabolic pathways converge on the TCA cycle. Reactions that form intermediates of the TCA cycle in order to replenish them (especially during the scarcity of the intermediates) are called anaplerotic reactions. The citric acid cycle is the third step in carbohydrate catabolism (the breakdown of sugars). Glycolysis breaks glucose (a six-carbon-molecule) down into pyruvate (a three-carbon molecule). In eukaryotes, pyruvate moves into the mitochondria. It is converted into acetyl-CoA by decarboxylation and enters the citric acid cycle. In protein catabolism, proteins are broken down by proteases into their constituent amino acids. The carbon backbone of these amino acids can become a source of energy by being converted to acetyl-CoA and entering into the citric acid cycle. In fat catabolism, triglycerides are hydrolyzed to break them into fatty acids and glycerol. In the liver the glycerol can be converted into glucose via dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by way of gluconeogenesis. In many tissues, especially heart tissue, fatty acids are broken down through a process known as beta oxidation, which results in acetyl-CoA, which can be used in the citric acid cycle. Beta oxidation of fatty acids with an odd number of methylene bridges produces propionyl CoA, which is then converted into succinyl-CoA and fed into the citric acid cycle.[] The total energy gained from the complete breakdown of one molecule of glucose by glycolysis, the citric acid cycle, and oxidative phosphorylation equals about 30 ATP molecules, in eukaryotes. The citric acid cycle is called an amphibolic pathway because it participates in both catabolism and anabolism.

Interactive pathway map Click on genes, proteins and metabolites below to link to respective articles. [2]

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Citric acid cycle

TCA Cycle edit [3] [2] The interactive pathway map can be edited at WikiPathways: [3] http:/ / www. wikipathways. org/ index. php/ Pathway:WP78

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Citric acid cycle

References External links • An animation of the citric acid cycle (http://www.science.smith.edu/departments/Biology/Bio231/krebs. html) at Smith College • Citric acid cycle variants (http://biocyc.org/META/NEW-IMAGE?object=TCA-VARIANTS) at MetaCyc • Pathways connected to the citric acid cycle (http://www.genome.ad.jp/kegg/pathway/map/map00020.html) at Kyoto Encyclopedia of Genes and Genomes • Introduction at Khan Academy (https://www.khanacademy.org/science/biology/cellular-respiration/v/ krebs---citric-acid-cycle)

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Oxidative phosphorylation Oxidative phosphorylation Oxidative phosphorylation (or OXPHOS in short) is the metabolic pathway in which the mitochondria in cells use their structure, enzymes, and energy released by the oxidation of nutrients to reform ATP. Although the many forms of life on earth use a range of different nutrients, ATP is the molecule that supplies energy to metabolism. Almost all aerobic organisms carry out oxidative phosphorylation. This pathway is probably so pervasive because it is a highly efficient way of releasing energy, compared to alternative fermentation processes such as anaerobic glycolysis. During oxidative phosphorylation, The electron transport chain in the mitochondrion is the site of oxidative phosphorylation electrons are transferred from electron in eukaryotes. The NADH and succinate generated in the citric acid cycle are oxidized, donors to electron acceptors such as releasing energy to power the ATP synthase. oxygen, in redox reactions. These redox reactions release energy, which is used to form ATP. In eukaryotes, these redox reactions are carried out by a series of protein complexes within the cell's intermembrane wall mitochondria, whereas, in prokaryotes, these proteins are located in the cells' intermembrane space. These linked sets of proteins are called electron transport chains. In eukaryotes, five main protein complexes are involved, whereas in prokaryotes many different enzymes are present, using a variety of electron donors and acceptors. The energy released by electrons flowing through this electron transport chain is used to transport protons across the inner mitochondrial membrane, in a process called electron transport. This generates potential energy in the form of a pH gradient and an electrical potential across this membrane. This store of energy is tapped by allowing protons to flow back across the membrane and down this gradient, through a large enzyme called ATP synthase; this process is known as chemiosmosis. This enzyme uses this energy to generate ATP from adenosine diphosphate (ADP), in a phosphorylation reaction. This reaction is driven by the proton flow, which forces the rotation of a part of the enzyme; the ATP synthase is a rotary mechanical motor. Although oxidative phosphorylation is a vital part of metabolism, it produces reactive oxygen species such as superoxide and hydrogen peroxide, which lead to propagation of free radicals, damaging cells and contributing to disease and, possibly, aging (senescence). The enzymes carrying out this metabolic pathway are also the target of many drugs and poisons that inhibit their activities.

Oxidative phosphorylation

234

Overview of energy transfer by chemiosmosis Oxidative phosphorylation works by using energy-releasing chemical reactions to drive energy-requiring reactions: The two sets of reactions are said to be coupled. This means one cannot occur without the other. The flow of electrons through the electron transport chain, from electron donors such as NADH to electron acceptors such as oxygen, is an exergonic process– it releases energy, whereas the synthesis of ATP is an endergonic process, which requires an input of energy. Both the electron transport chain and the ATP synthase are embedded in a membrane, and energy is transferred from electron transport chain to the ATP synthase by movements of protons across this membrane, in a process called chemiosmosis.[1] In practice, this is like a simple electric circuit, with a current of protons being driven from the negative N-side of the membrane to the positive P-side by the proton-pumping enzymes of the electron transport chain. These enzymes are like a battery, as they perform work to drive current through the circuit. The movement of protons creates an electrochemical gradient across the membrane, which is often called the proton-motive force. It has two components: a difference in proton concentration (a H+ gradient, ΔpH) and a difference in electric potential, with the N-side having a negative charge.[] ATP synthase releases this stored energy by completing the circuit and allowing protons to flow down the electrochemical gradient, back to the N-side of the membrane.[] This kinetic energy drives the rotation of part of the enzymes structure and couples this motion to the synthesis of ATP. The two components of the proton-motive force are thermodynamically equivalent: In mitochondria, the largest part of energy is provided by the potential; in alkaliphile bacteria the electrical energy even has to compensate for a counteracting inverse pH difference. Inversely, chloroplasts operate mainly on ΔpH. However, they also require a small membrane potential for the kinetics of ATP synthesis. At least in the case of the fusobacterium P. modestum it drives the counter-rotation of subunits a and c of the FO motor of ATP synthase.[] The amount of energy released by oxidative phosphorylation is high, compared with the amount produced by anaerobic fermentation. Glycolysis produces only 2 ATP molecules, but somewhere between 30 and 36 ATPs are produced by the oxidative phosphorylation of the 10 NADH and 2 succinate molecules made by converting one molecule of glucose to carbon dioxide and water,[2] while each cycle of beta oxidation of a fatty acid yields about 14 ATPs. These ATP yields are theoretical maximum values; in practice, some protons leak across the membrane, lowering the yield of ATP.[3]

Electron and proton transfer molecules

Reduction of coenzyme Q from its ubiquinone form (Q) to the reduced ubiquinol form (QH2).

The electron transport chain carries both protons and electrons, passing electrons from donors to acceptors, and transporting protons across a membrane. These processes use both soluble and protein-bound transfer molecules. In mitochondria, electrons are transferred within the intermembrane space by the water-soluble electron transfer protein cytochrome c.[4] This carries only electrons, and these are transferred by the reduction and oxidation of an iron atom that the protein holds within a heme group in its structure. Cytochrome c is also found in some bacteria, where it is located within the periplasmic space.[5] Within the inner mitochondrial membrane, the lipid-soluble electron carrier coenzyme Q10 (Q) carries

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both electrons and protons by a redox cycle.[6] This small benzoquinone molecule is very hydrophobic, so it diffuses freely within the membrane. When Q accepts two electrons and two protons, it becomes reduced to the ubiquinol form (QH2); when QH2 releases two electrons and two protons, it becomes oxidized back to the ubiquinone (Q) form. As a result, if two enzymes are arranged so that Q is reduced on one side of the membrane and QH2 oxidized on the other, ubiquinone will couple these reactions and shuttle protons across the membrane.[7] Some bacterial electron transport chains use different quinones, such as menaquinone, in addition to ubiquinone.[] Within proteins, electrons are transferred between flavin cofactors,[][] iron–sulfur clusters, and cytochromes. There are several types of iron–sulfur cluster. The simplest kind found in the electron transfer chain consists of two iron atoms joined by two atoms of inorganic sulfur; these are called [2Fe–2S] clusters. The second kind, called [4Fe–4S], contains a cube of four iron atoms and four sulfur atoms. Each iron atom in these clusters is coordinated by an additional amino acid, usually by the sulfur atom of cysteine. Metal ion cofactors undergo redox reactions without binding or releasing protons, so in the electron transport chain they serve solely to transport electrons through proteins. Electrons move quite long distances through proteins by hopping along chains of these cofactors.[8] This occurs by quantum tunnelling, which is rapid over distances of less than 1.4×10−9 m.[9]

Eukaryotic electron transport chains Many catabolic biochemical processes, such as glycolysis, the citric acid cycle, and beta oxidation, produce the reduced coenzyme NADH. This coenzyme contains electrons that have a high transfer potential; in other words, they will release a large amount of energy upon oxidation. However, the cell does not release this energy all at once, as this would be an uncontrollable reaction. Instead, the electrons are removed from NADH and passed to oxygen through a series of enzymes that each release a small amount of the energy. This set of enzymes, consisting of complexes I through IV, is called the electron transport chain and is found in the inner membrane of the mitochondrion. Succinate is also oxidized by the electron transport chain, but feeds into the pathway at a different point. In eukaryotes, the enzymes in this electron transport system use the energy released from the oxidation of NADH to pump protons across the inner membrane of the mitochondrion. This causes protons to build up in the intermembrane space, and generates an electrochemical gradient across the membrane. The energy stored in this potential is then used by ATP synthase to produce ATP. Oxidative phosphorylation in the eukaryotic mitochondrion is the best-understood example of this process. The mitochondrion is present in almost all eukaryotes, with the exception of anaerobic protozoa such as Trichomonas vaginalis that instead reduce protons to hydrogen in a remnant mitochondrion called a hydrogenosome.[10]

Typical respiratory enzymes and substrates in eukaryotes. Respiratory enzyme

Redox pair

Midpoint potential (Volts)

NADH dehydrogenase

NAD+ / NADH

−0.32

Succinate dehydrogenase

FMN or FAD / FMNH2 or FADH2

−0.20

[11] [11]

[11]

Cytochrome bc1 complex Coenzyme Q10ox / Coenzyme Q10red

+0.06

Cytochrome bc1 complex

Cytochrome box / Cytochrome bred

+0.12

Complex IV

Cytochrome cox / Cytochrome cred

+0.22

Complex IV

Cytochrome aox / Cytochrome ared

+0.29

Complex IV

O2 / HO−

+0.82

Conditions: pH = 7

[11]

[11] [11] [11] [11]

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NADH-coenzyme Q oxidoreductase (complex I) NADH-coenzyme Q oxidoreductase, also known as NADH dehydrogenase or complex I, is the first protein in the electron transport chain.[] Complex I is a giant enzyme with the mammalian complex I having 46 subunits and a molecular mass of about 1,000 kilodaltons (kDa).[] The structure is known in detail only from a bacterium;[][12] in most organisms the complex resembles a boot with a large "ball" poking out from the membrane into the mitochondrion.[13][14] The genes that encode the individual proteins are contained in both the cell nucleus and the mitochondrial genome, as is the case for many enzymes present in the mitochondrion.

Complex I or NADH-Q oxidoreductase. The abbreviations are discussed in the text. In all diagrams of respiratory complexes in this article, the matrix is at the bottom, with the intermembrane space above.

The reaction that is catalyzed by this enzyme is the two electron oxidation of NADH by coenzyme Q10 or ubiquinone (represented as Q in the equation below), a lipid-soluble quinone that is found in the mitochondrion membrane:

The start of the reaction, and indeed of the entire electron chain, is the binding of a NADH molecule to complex I and the donation of two electrons. The electrons enter complex I via a prosthetic group attached to the complex, flavin mononucleotide (FMN). The addition of electrons to FMN converts it to its reduced form, FMNH2. The electrons are then transferred through a series of iron–sulfur clusters: the second kind of prosthetic group present in the complex.[] There are both [2Fe–2S] and [4Fe–4S] iron–sulfur clusters in complex I. As the electrons pass through this complex, four protons are pumped from the matrix into the intermembrane space. Exactly how this occurs is unclear, but it seems to involve conformational changes in complex I that cause the protein to bind protons on the N-side of the membrane and release them on the P-side of the membrane.[15] Finally, the electrons are transferred from the chain of iron–sulfur clusters to a ubiquinone molecule in the membrane.[] Reduction of ubiquinone also contributes to the generation of a proton gradient, as two protons are taken up from the matrix as it is reduced to ubiquinol (QH2).

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Succinate-Q oxidoreductase (complex II) Succinate-Q oxidoreductase, also known as complex II or succinate dehydrogenase, is a second entry point to the electron transport chain.[16] It is unusual because it is the only enzyme that is part of both the citric acid cycle and the electron transport chain. Complex II consists of four protein subunits and contains a bound flavin adenine dinucleotide (FAD) cofactor, iron–sulfur clusters, and a heme group that does not participate in electron transfer to coenzyme Q, but is believed to be important in decreasing production of reactive oxygen species.[17][18] It oxidizes succinate to fumarate and reduces ubiquinone. As this reaction releases less energy than the oxidation of NADH, complex II does not transport protons across the membrane and does not contribute to the proton gradient. Complex II: Succinate-Q oxidoreductase.

In some eukaryotes, such as the parasitic worm Ascaris suum, an enzyme similar to complex II, fumarate reductase (menaquinol:fumarate oxidoreductase, or QFR), operates in reverse to oxidize ubiquinol and reduce fumarate. This allows the worm to survive in the anaerobic environment of the large intestine, carrying out anaerobic oxidative phosphorylation with fumarate as the electron acceptor.[19] Another unconventional function of complex II is seen in the malaria parasite Plasmodium falciparum. Here, the reversed action of complex II as an oxidase is important in regenerating ubiquinol, which the parasite uses in an unusual form of pyrimidine biosynthesis.[20]

Electron transfer flavoprotein-Q oxidoreductase Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-Q oxidoreductase), also known as electron transferring-flavoprotein dehydrogenase, is a third entry point to the electron transport chain. It is an enzyme that accepts electrons from electron-transferring flavoprotein in the mitochondrial matrix, and uses these electrons to reduce ubiquinone.[21] This enzyme contains a flavin and a [4Fe–4S] cluster, but, unlike the other respiratory complexes, it attaches to the surface of the membrane and does not cross the lipid bilayer.[22]

In mammals, this metabolic pathway is important in beta oxidation of fatty acids and catabolism of amino acids and choline, as it accepts electrons from multiple acetyl-CoA dehydrogenases.[23][24] In plants, ETF-Q oxidoreductase is also important in the metabolic responses that allow survival in extended periods of darkness.[25]

Oxidative phosphorylation

Q-cytochrome c oxidoreductase (complex III) Q-cytochrome c oxidoreductase is also known as cytochrome c reductase, cytochrome bc1 complex, or simply complex III.[26][27] In mammals, this enzyme is a dimer, with each subunit complex containing 11 protein subunits, an [2Fe-2S] iron–sulfur cluster and three cytochromes: one cytochrome c1 and two b [28] cytochromes. A cytochrome is a kind of electron-transferring protein The two electron transfer steps in complex III: Q-cytochrome c oxidoreductase. After that contains at least one heme group. each step, Q (in the upper part of the figure) leaves the enzyme. The iron atoms inside complex III’s heme groups alternate between a reduced ferrous (+2) and oxidized ferric (+3) state as the electrons are transferred through the protein. The reaction catalyzed by complex III is the oxidation of one molecule of ubiquinol and the reduction of two molecules of cytochrome c, a heme protein loosely associated with the mitochondrion. Unlike coenzyme Q, which carries two electrons, cytochrome c carries only one electron.

As only one of the electrons can be transferred from the QH2 donor to a cytochrome c acceptor at a time, the reaction mechanism of complex III is more elaborate than those of the other respiratory complexes, and occurs in two steps called the Q cycle.[29] In the first step, the enzyme binds three substrates, first, QH2, which is then oxidized, with one electron being passed to the second substrate, cytochrome c. The two protons released from QH2 pass into the intermembrane space. The third substrate is Q, which accepts the second electron from the QH2 and is reduced to Q.-, which is the ubisemiquinone free radical. The first two substrates are released, but this ubisemiquinone intermediate remains bound. In the second step, a second molecule of QH2 is bound and again passes its first electron to a cytochrome c acceptor. The second electron is passed to the bound ubisemiquinone, reducing it to QH2 as it gains two protons from the mitochondrial matrix. This QH2 is then released from the enzyme.[30] As coenzyme Q is reduced to ubiquinol on the inner side of the membrane and oxidized to ubiquinone on the other, a net transfer of protons across the membrane occurs, adding to the proton gradient.[] The rather complex two-step mechanism by which this occurs is important, as it increases the efficiency of proton transfer. If, instead of the Q cycle, one molecule of QH2 were used to directly reduce two molecules of cytochrome c, the efficiency would be halved, with only one proton transferred per cytochrome c reduced.[]

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Cytochrome c oxidase (complex IV) Cytochrome c oxidase, also known as complex IV, is the final protein complex in the electron transport chain.[31] The mammalian enzyme has an extremely complicated structure and contains 13 subunits, two heme groups, as well as multiple metal ion cofactors– in all three atoms of copper, one of magnesium and one of zinc.[32] This enzyme mediates the final reaction in the electron transport chain and transfers electrons to oxygen, while pumping protons across the membrane.[33] The final electron acceptor oxygen, which is also called the terminal electron acceptor, is reduced to water in this step. Both the direct pumping of protons and the consumption of matrix protons in the reduction of oxygen contribute to the proton gradient. The reaction catalyzed is the oxidation of cytochrome c and the reduction of oxygen:

Complex IV: cytochrome c oxidase.

Alternative reductases and oxidases Many eukaryotic organisms have electron transport chains that differ from the much-studied mammalian enzymes described above. For example, plants have alternative NADH oxidases, which oxidize NADH in the cytosol rather than in the mitochondrial matrix, and pass these electrons to the ubiquinone pool.[34] These enzymes do not transport protons, and, therefore, reduce ubiquinone without altering the electrochemical gradient across the inner membrane.[35] Another example of a divergent electron transport chain is the alternative oxidase, which is found in plants, as well as some fungi, protists, and possibly some animals.[36][37] This enzyme transfers electrons directly from ubiquinol to oxygen.[38] The electron transport pathways produced by these alternative NADH and ubiquinone oxidases have lower ATP yields than the full pathway. The advantages produced by a shortened pathway are not entirely clear. However, the alternative oxidase is produced in response to stresses such as cold, reactive oxygen species, and infection by pathogens, as well as other factors that inhibit the full electron transport chain.[39][40] Alternative pathways might, therefore, enhance an organisms' resistance to injury, by reducing oxidative stress.[41]

Organization of complexes The original model for how the respiratory chain complexes are organized was that they diffuse freely and independently in the mitochondrial membrane.[] However, recent data suggest that the complexes might form higher-order structures called supercomplexes or "respirasomes."[42] In this model, the various complexes exist as organized sets of interacting enzymes.[43] These associations might allow channeling of substrates between the various enzyme complexes, increasing the rate and efficiency of electron transfer.[44] Within such mammalian supercomplexes, some components would be present in higher amounts than others, with some data suggesting a ratio between complexes I/II/III/IV and the ATP synthase of approximately 1:1:3:7:4.[45] However, the debate over this supercomplex hypothesis is not completely resolved, as some data do not appear to fit with this model.[][46]

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Prokaryotic electron transport chains In contrast to the general similarity in structure and function of the electron transport chains in eukaryotes, bacteria and archaea possess a large variety of electron-transfer enzymes. These use an equally wide set of chemicals as substrates.[47] In common with eukaryotes, prokaryotic electron transport uses the energy released from the oxidation of a substrate to pump ions across a membrane and generate an electrochemical gradient. In the bacteria, oxidative phosphorylation in Escherichia coli is understood in most detail, while archaeal systems are at present poorly understood.[48] The main difference between eukaryotic and prokaryotic oxidative phosphorylation is that bacteria and archaea use many different substances to donate or accept electrons. This allows prokaryotes to grow under a wide variety of environmental conditions.[] In E. coli, for example, oxidative phosphorylation can be driven by a large number of pairs of reducing agents and oxidizing agents, which are listed below. The midpoint potential of a chemical measures how much energy is released when it is oxidized or reduced, with reducing agents having negative potentials and oxidizing agents positive potentials.

Respiratory enzymes and substrates in E. coli.[] Respiratory enzyme

Redox pair

Midpoint potential (Volts)

Formate dehydrogenase

Bicarbonate / Formate

−0.43

Hydrogenase

Proton / Hydrogen

−0.42

NADH dehydrogenase

NAD+ / NADH

−0.32

Glycerol-3-phosphate dehydrogenase DHAP / Gly-3-P

−0.19

Pyruvate oxidase

Acetate + Carbon dioxide / Pyruvate

Lactate dehydrogenase

Pyruvate / Lactate

D-amino acid dehydrogenase

2-oxoacid + ammonia / D-amino acid

Glucose dehydrogenase

Gluconate / Glucose

−0.14

Succinate dehydrogenase

Fumarate / Succinate

+0.03

Ubiquinol oxidase

Oxygen / Water

+0.82

Nitrate reductase

Nitrate / Nitrite

+0.42

Nitrite reductase

Nitrite / Ammonia

+0.36

Dimethyl sulfoxide reductase

DMSO / DMS

+0.16

Trimethylamine N-oxide reductase

TMAO / TMA

+0.13

Fumarate reductase

Fumarate / Succinate

+0.03

−0.19 ?

As shown above, E. coli can grow with reducing agents such as formate, hydrogen, or lactate as electron donors, and nitrate, DMSO, or oxygen as acceptors.[] The larger the difference in midpoint potential between an oxidizing and reducing agent, the more energy is released when they react. Out of these compounds, the succinate/fumarate pair is unusual, as its midpoint potential is close to zero. Succinate can therefore be oxidized to fumarate if a strong oxidizing agent such as oxygen is available, or fumarate can be reduced to succinate using a strong reducing agent such as formate. These alternative reactions are catalyzed by succinate dehydrogenase and fumarate reductase, respectively.[49] Some prokaryotes use redox pairs that have only a small difference in midpoint potential. For example, nitrifying bacteria such as Nitrobacter oxidize nitrite to nitrate, donating the electrons to oxygen. The small amount of energy released in this reaction is enough to pump protons and generate ATP, but not enough to produce NADH or NADPH directly for use in anabolism.[50] This problem is solved by using a nitrite oxidoreductase to produce enough

Oxidative phosphorylation proton-motive force to run part of the electron transport chain in reverse, causing complex I to generate NADH.[51][52] Prokaryotes control their use of these electron donors and acceptors by varying which enzymes are produced, in response to environmental conditions.[53] This flexibility is possible because different oxidases and reductases use the same ubiquinone pool. This allows many combinations of enzymes to function together, linked by the common ubiquinol intermediate.[] These respiratory chains therefore have a modular design, with easily interchangeable sets of enzyme systems. In addition to this metabolic diversity, prokaryotes also possess a range of isozymes– different enzymes that catalyze the same reaction. For example, in E. coli, there are two different types of ubiquinol oxidase using oxygen as an electron acceptor. Under highly aerobic conditions, the cell uses an oxidase with a low affinity for oxygen that can transport two protons per electron. However, if levels of oxygen fall, they switch to an oxidase that transfers only one proton per electron, but has a high affinity for oxygen.[54]

ATP synthase (complex V) ATP synthase, also called complex V, is the final enzyme in the oxidative phosphorylation pathway. This enzyme is found in all forms of life and functions in the same way in both prokaryotes and eukaryotes.[] The enzyme uses the energy stored in a proton gradient across a membrane to drive the synthesis of ATP from ADP and phosphate (Pi). Estimates of the number of protons required to synthesize one ATP have ranged from three to four,[55][56] with some suggesting cells can vary this ratio, to suit different conditions.[57]

This phosphorylation reaction is an equilibrium, which can be shifted by altering the proton-motive force. In the absence of a proton-motive force, the ATP synthase reaction will run from right to left, hydrolyzing ATP and pumping protons out of the matrix across the membrane. However, when the proton-motive force is high, the reaction is forced to run in the opposite direction; it proceeds from left to right, allowing protons to flow down their concentration gradient and turning ADP into ATP.[] Indeed, in the closely related vacuolar type H+-ATPases, the hydrolysis reaction is used to acidify cellular compartments, by pumping protons and hydrolysing ATP.[58] ATP synthase is a massive protein complex with a mushroom-like shape. The mammalian enzyme complex contains 16 subunits and has a mass of approximately 600 kilodaltons.[59] The portion embedded within the membrane is called FO and contains a ring of c subunits and the proton channel. The stalk and the ball-shaped headpiece is called F1 and is the site of ATP synthesis. The ball-shaped complex at the end of the F1 portion contains six proteins of two different kinds (three α subunits and three β subunits), whereas the "stalk" consists of one protein: the γ subunit, with the tip of the stalk extending into the ball of α and β subunits.[60] Both the α and β subunits bind nucleotides, but only the β subunits catalyze the ATP synthesis reaction. Reaching along the side of the F1 portion and back into the membrane is a long rod-like subunit that anchors the α and β subunits into the base of the enzyme. As protons cross the membrane through the channel in the base of ATP synthase, the FO proton-driven motor rotates.[61] Rotation might be caused by changes in the ionization of amino acids in the ring of c subunits causing electrostatic interactions that propel the ring of c subunits past the proton channel.[62] This rotating ring in turn drives the rotation of the central axle (the γ subunit stalk) within the α and β subunits. The α and β subunits are prevented from rotating themselves by the side-arm, which acts as a stator. This movement of the tip of the γ subunit within the ball of α and β subunits provides the energy for the active sites in the β subunits to undergo a cycle of movements that produces and then releases ATP.[]

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Oxidative phosphorylation

242

This ATP synthesis reaction is called the binding change mechanism and involves the active site of a β subunit cycling between three states.[] In the "open" state, ADP and phosphate enter the active site (shown in brown in the diagram). The protein then closes up around the molecules and binds them loosely– the "loose" state (shown in red). The enzyme then changes shape again and forces these molecules together, with the active site in the resulting "tight" state (shown in pink) binding the newly produced ATP molecule with very high affinity. Finally, the active site cycles back to the open state, releasing ATP and binding more ADP and phosphate, ready for the next cycle. Mechanism of ATP synthase. ATP is shown in red, ADP and

In some bacteria and archaea, ATP synthesis is driven by the phosphate in pink and the rotating γ subunit in black. movement of sodium ions through the cell membrane, rather than the movement of protons.[63][] Archaea such as Methanococcus also contain the A1Ao synthase, a form of the enzyme that contains additional proteins with little similarity in sequence to other bacterial and eukaryotic ATP synthase subunits. It is possible that, in some species, the A1Ao form of the enzyme is a specialized sodium-driven ATP synthase,[64] but this might not be true in all cases.[]

Reactive oxygen species Molecular oxygen is an ideal terminal electron acceptor because it is a strong oxidizing agent. The reduction of oxygen does involve potentially harmful intermediates.[] Although the transfer of four electrons and four protons reduces oxygen to water, which is harmless, transfer of one or two electrons produces superoxide or peroxide anions, which are dangerously reactive.

These reactive oxygen species and their reaction products, such as the hydroxyl radical, are very harmful to cells, as they oxidize proteins and cause mutations in DNA. This cellular damage might contribute to disease and is proposed as one cause of aging.[65][66] The cytochrome c oxidase complex is highly efficient at reducing oxygen to water, and it releases very few partly reduced intermediates; however small amounts of superoxide anion and peroxide are produced by the electron transport chain.[] Particularly important is the reduction of coenzyme Q in complex III, as a highly reactive ubisemiquinone free radical is formed as an intermediate in the Q cycle. This unstable species can lead to electron "leakage" when electrons transfer directly to oxygen, forming superoxide.[67] As the production of reactive oxygen species by these proton-pumping complexes is greatest at high membrane potentials, it has been proposed that mitochondria regulate their activity to maintain the membrane potential within a narrow range that balances ATP production against oxidant generation.[68] For instance, oxidants can activate uncoupling proteins that reduce membrane potential.[69] To counteract these reactive oxygen species, cells contain numerous antioxidant systems, including antioxidant vitamins such as vitamin C and vitamin E, and antioxidant enzymes such as superoxide dismutase, catalase, and peroxidases,[] which detoxify the reactive species, limiting damage to the cell.

Oxidative phosphorylation

243

Inhibitors There are several well-known drugs and toxins that inhibit oxidative phosphorylation. Although any one of these toxins inhibits only one enzyme in the electron transport chain, inhibition of any step in this process will halt the rest of the process. For example, if oligomycin inhibits ATP synthase, protons cannot pass back into the mitochondrion.[] As a result, the proton pumps are unable to operate, as the gradient becomes too strong for them to overcome. NADH is then no longer oxidized and the citric acid cycle ceases to operate because the concentration of NAD+ falls below the concentration that these enzymes can use. Compounds Cyanide Carbonmonoxide Azide Oligomycin

Use Poisons

Effect on oxidative phosphorylation Inhibit the electron transport chain by binding more strongly than oxygen to the Fe–Cu center in cytochrome c [70] oxidase, preventing the reduction of oxygen.

Antibiotic Inhibits ATP synthase by blocking the flow of protons through the F subunit.[] o

CCCP 2,4-Dinitrophenol

Poisons

Rotenone

Pesticide Prevents the transfer of electrons from complex I to ubiquinone by blocking the ubiquinone-binding site.[72]

Malonate and oxaloacetate

Ionophores that disrupt the proton gradient by carrying protons across a membrane. This ionophore uncouples [71] proton pumping from ATP synthesis because it carries protons across the inner mitochondrial membrane.

[73]

Competitive inhibitors of succinate dehydrogenase (complex II).

Not all inhibitors of oxidative phosphorylation are toxins. In brown adipose tissue, regulated proton channels called uncoupling proteins can uncouple respiration from ATP synthesis.[74] This rapid respiration produces heat, and is particularly important as a way of maintaining body temperature for hibernating animals, although these proteins may also have a more general function in cells' responses to stress.[75]

History The field of oxidative phosphorylation began with the report in 1906 by Arthur Harden of a vital role for phosphate in cellular fermentation, but initially only sugar phosphates were known to be involved.[76] However, in the early 1940s, the link between the oxidation of sugars and the generation of ATP was firmly established by Herman Kalckar,[77] confirming the central role of ATP in energy transfer that had been proposed by Fritz Albert Lipmann in 1941.[78] Later, in 1949, Morris Friedkin and Albert L. Lehninger proved that the coenzyme NADH linked metabolic pathways such as the citric acid cycle and the synthesis of ATP.[79] For another twenty years, the mechanism by which ATP is generated remained mysterious, with scientists searching for an elusive "high-energy intermediate" that would link oxidation and phosphorylation reactions.[80] This puzzle was solved by Peter D. Mitchell with the publication of the chemiosmotic theory in 1961.[81] At first, this proposal was highly controversial, but it was slowly accepted and Mitchell was awarded a Nobel prize in 1978.[82][83] Subsequent research concentrated on purifying and characterizing the enzymes involved, with major contributions being made by David E. Green on the complexes of the electron-transport chain, as well as Efraim Racker on the ATP synthase.[84] A critical step towards solving the mechanism of the ATP synthase was provided by Paul D. Boyer, by his development in 1973 of the "binding change" mechanism, followed by his radical proposal of rotational catalysis in 1982.[][85] More recent work has included structural studies on the enzymes involved in oxidative phosphorylation by John E. Walker, with Walker and Boyer being awarded a Nobel Prize in 1997.[86]

Oxidative phosphorylation

References [11] Medical CHEMISTRY Compendium. By Anders Overgaard Pedersen and Henning Nielsen. Aarhus University. 2008 [12] Efremov R.G., Baradaran R., & Sazanov L.A., (2010) The arcdhitecture of respiratory complex I, Nature 465, 441-445

Further reading Introductory • Nelson DL; Cox MM (2004). Lehninger Principles of Biochemistry (4th ed.). W. H. Freeman. ISBN0-7167-4339-6. • Schneider ED; Sagan D (2006). Into the Cool: Energy Flow, Thermodynamics and Life (1st ed.). University of Chicago Press. ISBN0-226-73937-6. • Lane N (2006). Power, Sex, Suicide: Mitochondria and the Meaning of Life (1st ed.). Oxford University Press, USA. ISBN0-19-920564-7. Advanced • Nicholls DG; Ferguson SJ (2002). Bioenergetics 3 (1st ed.). Academic Press. ISBN0-12-518121-3. • Haynie D (2001). Biological Thermodynamics (1st ed.). Cambridge University Press. ISBN0-521-79549-4. • Rajan SS (2003). Introduction to Bioenergetics (1st ed.). Anmol. ISBN81-261-1364-2. • Wikstrom M (Ed) (2005). Biophysical and Structural Aspects of Bioenergetics (1st ed.). Royal Society of Chemistry. ISBN0-85404-346-2.

External links General resources • Animated diagrams illustrating oxidative phosphorylation (http://www.wiley.com/legacy/college/boyer/ 0470003790/animations/electron_transport/electron_transport.htm) Wiley and Co Concepts in Biochemistry • ATP synthase - the rotary engine in the cell (http://www.res.titech.ac.jp/~seibutu/) Brief introduction, including videos of microscope images of the enzyme rotating, at Tokyo Institute of Technology • On-line biophysics lectures (http://www.life.uiuc.edu/crofts/bioph354/) Antony Crofts, University of Illinois at Urbana-Champaign Structural resources • Animations of the ATP synthase (http://nature.berkeley.edu/~hongwang/Project/ATP_synthase/) Hongyun Wang and George Oster, University of California, Berkeley • PDB molecule of the month: • ATP synthase (http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/ pdb72_1.html) • Cytochrome c (http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/ pdb36_1.html) • Cytochrome c oxidase (http://www.rcsb.org/pdb/static.do?p=education_discussion/ molecule_of_the_month/pdb5_1.html) • Interactive molecular models at Universidade Fernando Pessoa: • • • •

NADH dehydrogenase (http://www2.ufp.pt/~pedros/anim/2frame-ien.htm) succinate dehydrogenase (http://www2.ufp.pt/~pedros/anim/2frame-iien.htm) Coenzyme Q - cytochrome c reductase (http://www2.ufp.pt/~pedros/anim/2frame-iiien.htm) cytochrome c oxidase (http://www2.ufp.pt/~pedros/anim/2frame-iven.htm)

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Photosynthesis Photosynthesis Photosynthesis is a process used by plants and other autotrophic organisms to convert light energy, normally from the sun, into chemical energy that can be used to fuel the organisms' activities. Carbohydrates, such as sugars, are synthesized from carbon dioxide and water (hence the name photosynthesis, from the Greek φώτο[photo-], "light," and σύνθεσις [synthesis], "putting together") during the process. Oxygen is also released, mostly as a waste product. Most plants, most algae, and cyanobacteria perform the process of photosynthesis, and are called photoautotrophs. Photosynthesis maintains atmospheric oxygen levels and supplies most of the energy necessary for all life on Earth,[] except for chemotrophs, which gain energy through oxidative chemical reactions. Although photosynthesis is performed differently by different species, the process always begins when energy from light is absorbed by proteins called reaction centres that contain green chlorophyll pigments. In plants, these proteins are held inside organelles called chloroplasts, which are most abundant in leaf cells, while in bacteria they are embedded in the plasma membrane. In these light-dependent reactions, some energy is used to strip electrons from suitable substances such as water. This produces oxygen gas and hydrogen ions, which are transferred to a compound called nicotinamide adenine dinucleotide phosphate (NADP+), reducing it to NADPH. More light energy is transferred to chemical energy in the generation of adenosine triphosphate (ATP), the "energy currency" of cells. In plants, algae and cyanobacteria, sugars are produced by a sequence of light-independent reactions called the Calvin cycle, but some bacteria use different mechanisms, such as the reverse Krebs cycle. In the Calvin cycle, atmospheric carbon dioxide is incorporated into already existing organic carbon compounds, such as ribulose bisphosphate (RuBP).[] Using the ATP and NADPH produced by the light-dependent reactions, the resulting compounds are then reduced into triose phosphate. Of every six triose phosphate molecules produced, one is removed to form further carbohydrates and five are "recycled" back into the cycle to regenerate the original carbon dioxide acceptor, RuBP.

Schematic of photosynthesis in plants. The carbohydrates produced are stored in or used by the plant.

Overall equation for the type of photosynthesis that occurs in plants

Composite image showing the global distribution of photosynthesis, including both oceanic phytoplankton and terrestrial vegetation. Dark blue and green indicate regions of high photosynthetic activity in ocean and land respectively.

Photosynthesis The first photosynthetic organisms probably evolved early in the evolutionary history of life and most likely used reducing agents such as hydrogen or hydrogen sulfide as sources of electrons, rather than water.[] Cyanobacteria appeared later, and the excess oxygen they produced contributed to the oxygen catastrophe,[] which rendered the evolution of complex life possible. Today, the average rate of energy capture by photosynthesis globally is approximately 130terawatts,[][][] which is about six times larger than the current power consumption of human civilization.[] Photosynthetic organisms also convert around 100–115 thousand million metric tons (i.e., 100–115petagrams) of carbon into biomass per year.[][]

Overview Photosynthetic organisms are photoautotrophs, which means that they are able to synthesize food directly from carbon dioxide and water using energy from light. However, not all organisms that use light as a source of energy carry out photosynthesis, since photoheterotrophs use organic compounds, rather than carbon dioxide, as a source of carbon.[] In plants, algae and cyanobacteria, photosynthesis releases oxygen. This is called oxygenic photosynthesis. Although there are some differences between oxygenic photosynthesis in plants, algae, and cyanobacteria, the overall process is quite similar in these organisms. However, there are some types of bacteria that carry out anoxygenic photosynthesis, which consumes carbon dioxide but does not release oxygen. Carbon dioxide is converted into sugars in a process called carbon fixation. Carbon fixation is an endothermic redox reaction, so Photosynthesis changes sunlight into chemical photosynthesis needs to supply both a source of energy to drive this energy, splits water to liberate O2, and fixes CO2 into sugar. process, and the electrons needed to convert carbon dioxide into a carbohydrate. This addition of the electrons is a reduction reaction. In general outline and in effect, photosynthesis is the opposite of cellular respiration, in which glucose and other compounds are oxidized to produce carbon dioxide and water, and to release exothermic chemical energy to drive the organism's metabolism. However, the two processes take place through a different sequence of chemical reactions and in different cellular compartments. The general equation for photosynthesis is therefore: 2n CO2 + 2n DH2 + photons → 2(CH2O)n + 2n DO Carbon dioxide + electron donor + light energy → carbohydrate + oxidized electron donor In oxygenic photosynthesis water is the electron donor and, since its hydrolysis releases oxygen, the equation for this process is: 2n CO2 + 4n H2O + photons → 2(CH2O)n + 2n O2 + 2n H2O carbon dioxide + water + light energy → carbohydrate + oxygen + water Often 2n water molecules are cancelled on both sides, yielding: 2n CO2 + 2n H2O + photons → 2(CH2O)n + 2n O2 carbon dioxide + water + light energy → carbohydrate + oxygen Other processes substitute other compounds (such as arsenite) for water in the electron-supply role; for example some microbes use sunlight to oxidize arsenite to arsenate:[1] The equation for this reaction is: CO2 + (AsO33–) + photons → (AsO43–) + CO[]

246

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247

carbon dioxide + arsenite + light energy → arsenate + carbon monoxide (used to build other compounds in subsequent reactions) Photosynthesis occurs in two stages. In the first stage, light-dependent reactions or light reactions capture the energy of light and use it to make the energy-storage molecules ATP and NADPH. During the second stage, the light-independent reactions use these products to capture and reduce carbon dioxide. Most organisms that utilize photosynthesis to produce oxygen use visible light to do so, although at least three use shortwave infrared or, more specifically, far-red radiation.[2]

Photosynthetic membranes and organelles In photosynthetic bacteria, the proteins that gather light for photosynthesis are embedded within cell membranes, which is the simplest configuration these proteins are arranged.[] However, this membrane may be tightly folded into cylindrical sheets called thylakoids,[] or bunched up into round vesicles called intracytoplasmic membranes.[] These structures can fill most of the interior of a cell, giving the membrane a very large surface area and therefore increasing the amount of light that the bacteria can absorb.[]

Chloroplast ultrastructure: 1. outer membrane 2. intermembrane space3. inner membrane (1+2+3: envelope) 4. stroma (aqueous fluid) 5. thylakoid lumen (inside of thylakoid) 6. thylakoid membrane 7. granum (stack of thylakoids) 8. thylakoid (lamella) 9. starch 10. ribosome 11. plastidial DNA 12. plastoglobule (drop of lipids)

In plants and algae, photosynthesis takes place in organelles called chloroplasts. A typical plant cell contains about 10 to 100 chloroplasts. The chloroplast is enclosed by a membrane. This membrane is composed of a phospholipid inner membrane, a phospholipid outer membrane, and an intermembrane space between them. Within the membrane is an aqueous fluid called the stroma. The stroma contains stacks (grana) of thylakoids, which are the site of photosynthesis. The thylakoids are flattened disks, bounded by a membrane with a lumen or thylakoid space within it. The site of photosynthesis is the thylakoid membrane, which contains integral and peripheral membrane protein complexes, including the pigments that absorb light energy, which form the photosystems. Plants absorb light primarily using the pigment chlorophyll, which is the reason that most plants have a green color. Besides chlorophyll, plants also use pigments such as carotenes and xanthophylls.[] Algae also use chlorophyll, but various other pigments are present as phycocyanin, carotenes, and xanthophylls in green algae, phycoerythrin in red algae (rhodophytes) and fucoxanthin in brown algae and diatoms resulting in a wide variety of colors. These pigments are embedded in plants and algae in special antenna-proteins. In such proteins all the pigments are ordered to work well together. Such a protein is also called a light-harvesting complex. Although all cells in the green parts of a plant have chloroplasts, most of the energy is captured in the leaves, except in certain species adapted to conditions of strong sunlight and aridity, such as many Euphorbia and Cactus species, whose main photosynthetic organs are their stems. The cells in the interior tissues of a leaf, called the mesophyll, can contain between 450,000 and 800,000 chloroplasts for every square millimeter of leaf. The surface of the leaf is uniformly coated with a water-resistant waxy cuticle that protects the leaf from excessive evaporation of water and decreases the absorption of ultraviolet or blue light to reduce heating. The transparent epidermis layer allows light to pass through to the palisade mesophyll cells where most of the photosynthesis takes place.

Photosynthesis

248

Light reactions In the light reactions, one molecule of the pigment chlorophyll absorbs one photon and loses one electron. This electron is passed to a modified form of chlorophyll called pheophytin, which passes the electron to a quinone molecule, allowing the start of a flow of electrons down an electron transport chain that leads to the ultimate reduction of NADP to NADPH. In addition, this creates a proton gradient across the chloroplast membrane; its Light-dependent reactions of photosynthesis at the thylakoid membrane dissipation is used by ATP synthase for the concomitant synthesis of ATP. The chlorophyll molecule regains the lost electron from a water molecule through a process called photolysis, which releases a dioxygen (O2) molecule. The overall equation for the light-dependent reactions under the conditions of non-cyclic electron flow in green plants is:[] 2 H2O + 2 NADP+ + 3 ADP + 3 Pi + light → 2 NADPH + 2 H+ + 3 ATP + O2 Not all wavelengths of light can support photosynthesis. The photosynthetic action spectrum depends on the type of accessory pigments present. For example, in green plants, the action spectrum resembles the absorption spectrum for chlorophylls and carotenoids with peaks for violet-blue and red light. In red algae, the action spectrum overlaps with the absorption spectrum of phycobilins for red blue-green light, which allows these algae to grow in deeper waters that filter out the longer wavelengths used by green plants. The non-absorbed part of the light spectrum is what gives photosynthetic organisms their color (e.g., green plants, red algae, purple bacteria) and is the least effective for photosynthesis in the respective organisms.

Z scheme

The "Z scheme"

In plants, light-dependent reactions occur in the thylakoid membranes of the chloroplasts and use light energy to synthesize ATP and NADPH. The light-dependent reaction has two forms: cyclic and non-cyclic. In the non-cyclic reaction, the photons are captured in the light-harvesting antenna complexes of photosystem II by chlorophyll and other accessory pigments (see diagram at right). When a chlorophyll molecule at the core of the photosystem II reaction center obtains sufficient excitation energy from the adjacent antenna pigments, an electron is transferred to

Photosynthesis the primary electron-acceptor molecule, pheophytin, through a process called photoinduced charge separation. These electrons are shuttled through an electron transport chain, the so-called Z-scheme shown in the diagram, that initially functions to generate a chemiosmotic potential across the membrane. An ATP synthase enzyme uses the chemiosmotic potential to make ATP during photophosphorylation, whereas NADPH is a product of the terminal redox reaction in the Z-scheme. The electron enters a chlorophyll molecule in Photosystem I. The electron is excited due to the light absorbed by the photosystem. A second electron carrier accepts the electron, which again is passed down lowering energies of electron acceptors. The energy created by the electron acceptors is used to move hydrogen ions across the thylakoid membrane into the lumen. The electron is used to reduce the co-enzyme NADP, which has functions in the light-independent reaction. The cyclic reaction is similar to that of the non-cyclic, but differs in the form that it generates only ATP, and no reduced NADP (NADPH) is created. The cyclic reaction takes place only at photosystem I. Once the electron is displaced from the photosystem, the electron is passed down the electron acceptor molecules and returns to photosystem I, from where it was emitted, hence the name cyclic reaction.

Water photolysis The NADPH is the main reducing agent in chloroplasts, providing a source of energetic electrons to other reactions. Its production leaves chlorophyll with a deficit of electrons (oxidized), which must be obtained from some other reducing agent. The excited electrons lost from chlorophyll in photosystem I are replaced from the electron transport chain by plastocyanin. However, since photosystem II includes the first steps of the Z-scheme, an external source of electrons is required to reduce its oxidized chlorophyll a molecules. The source of electrons in green-plant and cyanobacterial photosynthesis is water. Two water molecules are oxidized by four successive charge-separation reactions by photosystem II to yield a molecule of diatomic oxygen and four hydrogen ions; the electron yielded in each step is transferred to a redox-active tyrosine residue that then reduces the photoxidized paired-chlorophyll a species called P680 that serves as the primary (light-driven) electron donor in the photosystem II reaction center. The oxidation of water is catalyzed in photosystem II by a redox-active structure that contains four manganese ions and a calcium ion; this oxygen-evolving complex binds two water molecules and stores the four oxidizing equivalents that are required to drive the water-oxidizing reaction. Photosystem II is the only known biological enzyme that carries out this oxidation of water. The hydrogen ions contribute to the transmembrane chemiosmotic potential that leads to ATP synthesis. Oxygen is a waste product of light-dependent reactions, but the majority of organisms on Earth use oxygen for cellular respiration, including photosynthetic organisms.[][]

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Light-independent reactions Calvin cycle In the light-independent (or "dark") reactions, the enzyme RuBisCO captures CO2 from the atmosphere and in a process that requires the newly formed NADPH, called the Calvin-Benson Cycle, releases three-carbon sugars, which are later combined to form sucrose and starch. The overall equation for the light-independent reactions in green plants is:[]:128 3 CO2 + 9 ATP + 6 NADPH + 6 H+ → C3H6O3-phosphate + 9 ADP + 8 Pi + 6 NADP+ + 3 H2O To be more specific, carbon fixation produces an intermediate product, which is then converted to the final carbohydrate products. The carbon skeletons produced by photosynthesis are then variously used to form other organic compounds, such as the building material cellulose, as precursors for lipid and amino acid biosynthesis, or as a fuel in cellular respiration. The latter occurs not only in plants but also in animals when the energy from plants gets passed through a food chain. The fixation or reduction of carbon dioxide is a process in which carbon dioxide combines with a five-carbon sugar, ribulose 1,5-bisphosphate Overview of the Calvin cycle and carbon fixation (RuBP), to yield two molecules of a three-carbon compound, glycerate 3-phosphate (GP), also known as 3-phosphoglycerate (PGA). GP, in the presence of ATP and NADPH from the light-dependent stages, is reduced to glyceraldehyde 3-phosphate (G3P). This product is also referred to as 3-phosphoglyceraldehyde (PGAL) or even as triose phosphate. Triose is a 3-carbon sugar (see carbohydrates). Most (5 out of 6 molecules) of the G3P produced is used to regenerate RuBP so the process can continue (see Calvin-Benson cycle). The 1 out of 6 molecules of the triose phosphates not "recycled" often condense to form hexose phosphates, which ultimately yield sucrose, starch and cellulose. The sugars produced during carbon metabolism yield carbon skeletons that can be used for other metabolic reactions like the production of amino acids and lipids.

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Carbon concentrating mechanisms On land In hot and dry conditions, plants close their stomata to prevent the loss of water. Under these conditions, CO2 will decrease, and oxygen gas, produced by the light reactions of photosynthesis, will decrease in the stem, not leaves, causing an increase of photorespiration by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and decrease in carbon fixation. Some plants have evolved mechanisms to increase the CO2 concentration in the leaves under these conditions. C4 plants chemically fix carbon dioxide in the cells of the mesophyll by adding it to the three-carbon molecule phosphoenolpyruvate (PEP), a reaction catalyzed by an enzyme called PEP carboxylase, creating the four-carbon organic acid oxaloacetic acid. Oxaloacetic acid or malate synthesized by this process is then translocated to specialized bundle sheath cells where the enzyme RuBisCO and other Calvin cycle enzymes are located, and where CO2 released by decarboxylation of the four-carbon acids is then fixed by RuBisCO activity to the three-carbon sugar 3-phosphoglyceric acids. The physical separation of RuBisCO from the Overview of C4 carbon fixation oxygen-generating light reactions reduces photorespiration and increases CO2 fixation and, thus, photosynthetic capacity of the leaf.[] C4 plants can produce more sugar than C3 plants in conditions of high light and temperature. Many important crop plants are C4 plants, including maize, sorghum, sugarcane, and millet. Plants that do not use PEP-carboxylase in carbon fixation are called C3 plants because the primary carboxylation reaction, catalyzed by RuBisCO, produces the three-carbon sugar 3-phosphoglyceric acids directly in the Calvin-Benson cycle. Over 90% of plants use C3 carbon fixation, compared to 3% that use C4 carbon fixation.[] Xerophytes, such as cacti and most succulents, also use PEP carboxylase to capture carbon dioxide in a process called Crassulacean acid metabolism (CAM). In contrast to C4 metabolism, which physically separates the CO2 fixation to PEP from the Calvin cycle, CAM temporally separates these two processes. CAM plants have a different leaf anatomy from C3 plants, and fix the CO2 at night, when their stomata are open. CAM plants store the CO2 mostly in the form of malic acid via carboxylation of phosphoenolpyruvate to oxaloacetate, which is then reduced to malate. Decarboxylation of malate during the day releases CO2 inside the leaves, thus allowing carbon fixation to 3-phosphoglycerate by RuBisCO. Sixteen thousand species of plants use CAM.[]

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In water Cyanobacteria possess carboxysomes, which increase the concentration of CO2 around RuBisCO to increase the rate of photosynthesis. An enzyme, carbonic anhydrase, located within the carboxysome releases CO2 from the dissolved hydrocarbonate ions (HCO3–). Before the CO2 diffuses out it is quickly sponged up by RuBisCO, which is concentrated within the carboxysomes. HCO3– ions are made from CO2 outside the cell by another carbonic anhydrase and are actively pumped into the cell by a membrane protein. They cannot cross the membrane as they are charged, and within the cytosol they turn back into CO2 very slowly without the help of carbonic anhydrase. This causes the HCO3– ions to accumulate within the cell from where they diffuse into the carboxysomes.[3] Pyrenoids in algae and hornworts also act to concentrate CO2 around rubisco.[4]

Order and kinetics The overall process of photosynthesis takes place in four stages:[] Stage

Description

Time scale

Energy transfer in antenna chlorophyll (thylakoid membranes)

femtosecond to picosecond

Transfer of electrons in photochemical reactions (thylakoid membranes) picosecond to nanosecond

Electron transport chain and ATP synthesis (thylakoid membranes)

microsecond to millisecond

Carbon fixation and export of stable products

millisecond to second

Efficiency Plants usually convert light into chemical energy with a photosynthetic efficiency of 3–6%.[5] Actual plants' photosynthetic efficiency varies with the frequency of the light being converted, light intensity, temperature and proportion of carbon dioxide in the atmosphere, and can vary from 0.1% to 8%.[6] By comparison, solar panels convert light into electric energy at an efficiency of approximately 6–20% for mass-produced panels, and above 40% in laboratory devices. Photosynthesis measurement systems are not designed to directly measure the amount of light absorbed by the leaf. Nevertheless, the light response curves that systems like the LCpro-SD produce, do allow comparisons in photosynthetic efficiency between plants.

Measuring the photosynthetic efficiency of wheat in the field using an LCpro-SD

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Evolution Early photosynthetic systems, such as those from green and purple sulfur and green and purple nonsulfur bacteria, are thought to have been anoxygenic, using various molecules as electron donors. Green and purple sulfur bacteria are thought to have used hydrogen and sulfur as an electron donor. Green nonsulfur bacteria used various amino and other organic acids. Purple nonsulfur bacteria used a variety of nonspecific organic molecules. The use of these molecules is consistent with the geological evidence that the atmosphere was highly reduced at that time.[citation needed] Fossils of what are thought to be filamentous photosynthetic organisms have been dated at 3.4 billion years old.[7][8]

Plant cells with visible chloroplasts (from a moss, Plagiomnium affine)

The main source of oxygen in the atmosphere is oxygenic photosynthesis, and its first appearance is sometimes referred to as the oxygen catastrophe. Geological evidence suggests that oxygenic photosynthesis, such as that in cyanobacteria, became important during the Paleoproterozoic era around 2 billion years ago. Modern photosynthesis in plants and most photosynthetic prokaryotes is oxygenic. Oxygenic photosynthesis uses water as an electron donor, which is oxidized to molecular oxygen (O2) in the photosynthetic reaction center.

Symbiosis and the origin of chloroplasts Several groups of animals have formed symbiotic relationships with photosynthetic algae. These are most common in corals, sponges and sea anemones. It is presumed that this is due to the particularly simple body plans and large surface areas of these animals compared to their volumes.[9] In addition, a few marine mollusks Elysia viridis and Elysia chlorotica also maintain a symbiotic relationship with chloroplasts they capture from the algae in their diet and then store in their bodies. This allows the mollusks to survive solely by photosynthesis for several months at a time.[][] Some of the genes from the plant cell nucleus have even been transferred to the slugs, so that the chloroplasts can be supplied with proteins that they need to survive.[] An even closer form of symbiosis may explain the origin of chloroplasts. Chloroplasts have many similarities with photosynthetic bacteria, including a circular chromosome, prokaryotic-type ribosomes, and similar proteins in the photosynthetic reaction center.[][] The endosymbiotic theory suggests that photosynthetic bacteria were acquired (by endocytosis) by early eukaryotic cells to form the first plant cells. Therefore, chloroplasts may be photosynthetic bacteria that adapted to life inside plant cells. Like mitochondria, chloroplasts still possess their own DNA, separate from the nuclear DNA of their plant host cells and the genes in this chloroplast DNA resemble those in cyanobacteria.[] DNA in chloroplasts codes for redox proteins such as photosynthetic reaction centers. The CoRR Hypothesis proposes that this Co-location is required for Redox Regulation.

Photosynthesis

Cyanobacteria and the evolution of photosynthesis The biochemical capacity to use water as the source for electrons in photosynthesis evolved once, in a common ancestor of extant cyanobacteria. The geological record indicates that this transforming event took place early in Earth's history, at least 2450–2320 million years ago (Ma), and, it is speculated, much earlier.[10] Available evidence from geobiological studies of Archean (>2500 Ma) sedimentary rocks indicates that life existed 3500 Ma, but the question of when oxygenic photosynthesis evolved is still unanswered. A clear paleontological window on cyanobacterial evolution opened about 2000 Ma, revealing an already-diverse biota of blue-greens. Cyanobacteria remained principal primary producers throughout the Proterozoic Eon (2500–543 Ma), in part because the redox structure of the oceans favored photoautotrophs capable of nitrogen fixation.[citation needed] Green algae joined blue-greens as major primary producers on continental shelves near the end of the Proterozoic, but only with the Mesozoic (251–65 Ma) radiations of dinoflagellates, coccolithophorids, and diatoms did primary production in marine shelf waters take modern form. Cyanobacteria remain critical to marine ecosystems as primary producers in oceanic gyres, as agents of biological nitrogen fixation, and, in modified form, as the plastids of marine algae.[] A 2010 study by researchers at Tel Aviv University discovered that the Oriental hornet (Vespa orientalis) converts sunlight into electric power using a pigment called xanthopterin. This is the first scientific evidence of a member of the animal kingdom engaging in photosynthesis.[11]

Discovery Although some of the steps in photosynthesis are still not completely understood, the overall photosynthetic equation has been known since the 19th century. Jan van Helmont began the research of the process in the mid-17th century when he carefully measured the mass of the soil used by a plant and the mass of the plant as it grew. After noticing that the soil mass changed very little, he hypothesized that the mass of the growing plant must come from the water, the only substance he added to the potted plant. His hypothesis was partially accurate — much of the gained mass also comes from carbon dioxide as well as water. However, this was a signaling point to the idea that the bulk of a plant's biomass comes from the inputs of photosynthesis, not the soil itself. Joseph Priestley, a chemist and minister, discovered that, when he isolated a volume of air under an inverted jar, and burned a candle in it, the candle would burn out very quickly, much before it ran out of wax. He further discovered that a mouse could similarly "injure" air. He then showed that the air that had been "injured" by the candle and the mouse could be restored by a plant. In 1778, Jan Ingenhousz, court physician to the Austrian Empress, repeated Priestley's experiments. He discovered that it was the influence of sunlight on the plant that could cause it to revive a mouse in a matter of hours. In 1796, Jean Senebier, a Swiss pastor, botanist, and naturalist, demonstrated that green plants consume carbon dioxide and release oxygen under the influence of light. Soon afterward, Nicolas-Théodore de Saussure showed that the increase in mass of the plant as it grows could not be due only to uptake of CO2 but also to the incorporation of water. Thus, the basic reaction by which photosynthesis is used to produce food (such as glucose) was outlined. Cornelis Van Niel made key discoveries explaining the chemistry of photosynthesis. By studying purple sulfur bacteria and green bacteria he was the first scientist to demonstrate that photosynthesis is a light-dependent redox reaction, in which hydrogen reduces carbon dioxide. Robert Emerson discovered two light reactions by testing plant productivity using different wavelengths of light. With the red alone, the light reactions were suppressed. When blue and red were combined, the output was much more substantial. Thus, there were two photosystems, one absorbing up to 600nm wavelengths, the other up to 700nm. The former is known as PSII, the latter is PSI. PSI contains only chlorophyll a, PSII contains primarily chlorophyll a with most of the available chlorophyll b, among other pigment. These include phycobilins, which are the red and blue pigments of red and blue algae respectively, and fucoxanthol for brown algae and diatoms. The

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process is most productive when absorption of quanta are equal in both the PSII and PSI, assuring that input energy from the antenna complex is divided between the PSI and PSII system, which in turn powers the photochemistry.[] Robert Hill thought that a complex of reactions consisting of an intermediate to cytochrome b6 (now a plastoquinone), another is from cytochrome f to a step in the carbohydrate-generating mechanisms. These are linked by plastoquinone, which does require energy to reduce cytochrome f for it is a sufficient reductant. Further experiments to prove that the oxygen developed during the photosynthesis of green plants came from water, were performed by Hill in 1937 and 1939. He showed that isolated chloroplasts give off oxygen in the presence of unnatural reducing agents like iron oxalate, ferricyanide or benzoquinone after exposure to light. The Hill reaction is as follows: 2 H2O + 2 A + (light, chloroplasts) → 2 AH2 + O2 where A is the electron acceptor. Therefore, in light, the electron acceptor is reduced and oxygen is evolved. Samuel Ruben and Martin Kamen used radioactive isotopes to determine that the oxygen liberated in photosynthesis came from the water.

Melvin Calvin works in his photosynthesis laboratory.

Melvin Calvin and Andrew Benson, along with James Bassham, elucidated the path of carbon assimilation (the photosynthetic carbon reduction cycle) in plants. The carbon reduction cycle is known as the Calvin cycle, which ignores the contribution of Bassham and Benson. Many scientists refer to the cycle as the Calvin-Benson Cycle, Benson-Calvin, and some even call it the Calvin-Benson-Bassham (or CBB) Cycle. Nobel Prize-winning scientist Rudolph A. Marcus was able to discover the function and significance of the electron transport chain. Otto Heinrich Warburg and Dean Burk discovered the I-quantum photosynthesis reaction that splits the CO2, activated by the respiration.[12] Louis N.M. Duysens and Jan Amesz discovered that chlorophyll a will absorb one light, oxidize cytochrome f, chlorophyll a (and other pigments) will absorb another light, but will reduce this same oxidized cytochrome, stating the two light reactions are in series.

Factors There are three main factors affecting photosynthesis and several corollary factors. The three main are: • Light irradiance and wavelength • Carbon dioxide concentration • Temperature.

Light intensity (irradiance), wavelength and temperature In the early 20th century, Frederick Blackman and Gabrielle Matthaei investigated the effects of light intensity (irradiance) and temperature on the rate of carbon assimilation.

The leaf is the primary site of photosynthesis in plants.

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• At constant temperature, the rate of carbon assimilation varies with irradiance, initially increasing as the irradiance increases. However, at higher irradiance, this relationship no longer holds and the rate of carbon assimilation reaches a plateau. • At constant irradiance, the rate of carbon assimilation increases as the temperature is increased over a limited range. This effect is seen only at high irradiance levels. At low irradiance, increasing the temperature has little influence on the rate of carbon assimilation. These two experiments illustrate vital points: First, from research it is known that, in general, photochemical reactions are not affected by temperature. However, these experiments clearly show that temperature affects the rate of carbon assimilation, so there must be two sets of reactions in the full process of carbon assimilation. These are, of course, the light-dependent 'photochemical' stage and the light-independent, temperature-dependent stage. Second, Blackman's experiments illustrate the concept of limiting factors. Another limiting factor is the wavelength of light. Cyanobacteria, which reside several meters underwater, cannot receive the correct wavelengths required to cause photoinduced charge separation in conventional photosynthetic pigments. To combat this problem, a series of proteins with different pigments surround the reaction center. This unit is called a phycobilisome.

Carbon dioxide levels and photorespiration As carbon dioxide concentrations rise, the rate at which sugars are made by the light-independent reactions increases until limited by other factors. RuBisCO, the enzyme that captures carbon dioxide in the light-independent reactions, has a binding affinity for both carbon dioxide and oxygen. When the concentration of carbon dioxide is high, RuBisCO will fix carbon dioxide. However, if the carbon dioxide Photorespiration concentration is low, RuBisCO will bind oxygen instead of carbon dioxide. This process, called photorespiration, uses energy, but does not produce sugars. RuBisCO oxygenase activity is disadvantageous to plants for several reasons: 1. One product of oxygenase activity is phosphoglycolate (2 carbon) instead of 3-phosphoglycerate (3 carbon). Phosphoglycolate cannot be metabolized by the Calvin-Benson cycle and represents carbon lost from the cycle. A high oxygenase activity, therefore, drains the sugars that are required to recycle ribulose 5-bisphosphate and for the continuation of the Calvin-Benson cycle. 2. Phosphoglycolate is quickly metabolized to glycolate that is toxic to a plant at a high concentration; it inhibits photosynthesis. 3. Salvaging glycolate is an energetically expensive process that uses the glycolate pathway, and only 75% of the carbon is returned to the Calvin-Benson cycle as 3-phosphoglycerate. The reactions also produce ammonia (NH3), which is able to diffuse out of the plant, leading to a loss of nitrogen. A highly simplified summary is: 2 glycolate + ATP → 3-phosphoglycerate + carbon dioxide + ADP + NH3 The salvaging pathway for the products of RuBisCO oxygenase activity is more commonly known as photorespiration, since it is characterized by light-dependent oxygen consumption and the release of carbon dioxide.

Photosynthesis

References [1] [5] [6] [7]

Anaerobic Photosynthesis, Chemical & Engineering News, 86, 33, August 18, 2008, p. 36 Chapter 1 – Biological energy production"> Govindjee, What is photosynthesis? (http:/ / www. life. uiuc. edu/ govindjee/ whatisit. htm) Photosynthesis got a really early start (http:/ / www. newscientist. com/ article/ mg18424671. 600-photosynthesis-got-a-really-early-start. html), New Scientist, 2 October 2004 [8] Revealing the dawn of photosynthesis (http:/ / www. newscientist. com/ article/ mg19125654. 200-revealing-the-dawn-of-photosynthesis. html), New Scientist, 19 August 2006 [12] Otto Warburg – Biography (http:/ / nobelprize. org/ nobel_prizes/ medicine/ laureates/ 1931/ warburg. html). Nobelprize.org (1970-08-01). Retrieved on 2011-11-03.

Further reading Books • Asimov, Isaac (1968). Photosynthesis. New York, London: Basic Books, Inc. ISBN0-465-05703-9. • Bidlack JE; Stern KR, Jansky S (2003). Introductory plant biology. New York: McGraw-Hill. ISBN0-07-290941-2. • Blankenship RE (2008). Molecular Mechanisms of Photosynthesis (2nd ed.). John Wiley & Sons Inc. ISBN0-470-71451-4. • Govindjee (1975). Bioenergetics of photosynthesis. Boston: Academic Press. ISBN0-12-294350-3. • Govindjee Beatty JT,Gest H, Allen JF (2006). Discoveries in Photosynthesis. Advances in Photosynthesis and Respiration 20. Berlin: Springer. ISBN1-4020-3323-0. • Gregory RL (1971). Biochemistry of photosynthesis. New York: Wiley-Interscience. ISBN0-471-32675-5. • Rabinowitch E, Govindjee (1969). Photosynthesis. London: J. Wiley. ISBN0-471-70424-5. • Reece, J, Campbell, N (2005). Biology. San Francisco: Pearson, Benjamin Cummings. ISBN0-8053-7146-X.

Papers • Gupta RS, Mukhtar T, Singh B (June 1999). "Evolutionary relationships among photosynthetic prokaryotes (Heliobacterium chlorum, Chloroflexus aurantiacus, cyanobacteria, Chlorobium tepidum and proteobacteria): implications regarding the origin of photosynthesis". Mol. Microbiol. 32 (5): 893–906. PMID 10361294 (http:// www.ncbi.nlm.nih.gov/pubmed/10361294). "implications regarding the origin of photosynthesis" • Blankenship RE (1992). "Origin and early evolution of photosynthesis". Photosyn. Res. 33: 91–111. PMID 11538390 (http://www.ncbi.nlm.nih.gov/pubmed/11538390). • Rutherford AW, Faller P (January 2003). "Photosystem II: evolutionary perspectives" (http://www.ncbi.nlm. nih.gov/pmc/articles/PMC1693113). Philos. Trans. R. Soc. Lond., B, Biol. Sci. 358 (1429): 245–53. doi: 10.1098/rstb.2002.1186 (http://dx.doi.org/10.1098/rstb.2002.1186). PMC 1693113 (http://www.ncbi.nlm. nih.gov/pmc/articles/PMC1693113). PMID 12594932 (http://www.ncbi.nlm.nih.gov/pubmed/12594932).

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External links • A collection of photosynthesis pages for all levels from a renowned expert (Govindjee) (http://www.life.uiuc. edu/govindjee/linksPSed.htm) • In depth, advanced treatment of photosynthesis, also from Govindjee (http://www.life.uiuc.edu/govindjee/ paper/gov.html) • Science Aid: Photosynthesis (http://scienceaid.co.uk/biology/biochemistry/photosynthesis.html) Article appropriate for high school science • Metabolism, Cellular Respiration and Photosynthesis – The Virtual Library of Biochemistry and Cell Biology (http://www.biochemweb.org/metabolism.shtml) • Overall examination of Photosynthesis at an intermediate level (http://www.chemsoc.org/networks/learnnet/ cfb/Photosynthesis.htm) • Overall Energetics of Photosynthesis (http://www.life.uiuc.edu/govindjee/photosynBook.html) • Photosynthesis Discovery Milestones (http://www.juliantrubin.com/bigten/photosynthesisexperiments.html) – experiments and background • The source of oxygen produced by photosynthesis (http://bcs.whfreeman.com/thelifewire/content/chp08/ 0802001.html) Interactive animation, a textbook tutorial • Jessica Marshall (2011-03-29). "First practical artificial leaf makes debut" (http://news.discovery.com/earth/ artificial-leaf-technology-solar-110329.html). Discovery News. • Photosynthesis – Light Dependent & Light Independent Stages (http://www.biology-innovation.co.uk/pages/ plant-biology-ecology/photosynthesis/) • Khan Academy, video introduction (http://www.khanacademy.org/video/photosynthesis?playlist=Biology)

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Lipid metabolism Fatty acid synthesis Fatty acid synthesis is the creation of fatty acids from acetyl-CoA and malonyl-CoA precursors through action of enzymes called fatty acid synthases. It is an important part of the lipogenesis process, which - together with glycolysis - stands behind creating fats from blood sugar in living organisms.

Straight-Chain Fatty Acids Straight-chain fatty acids occur in two types; saturated and unsaturated.

Saturated Straight-Chain Fatty Acids Much like β-oxidation, straight-chain fatty acid synthesis occurs via the six recurring reactions shown below, until the 16-carbon palmitic acid is produced.[1] The diagrams presented show how fatty acids are synthesized in microorganisms and list the enzymes found in Escherichia coli.[1] These reactions are performed by fatty acid synthase II (FASII), which in general contain multiple enzymes that act as one complex. FASII is present in prokaryotes, plants, fungi, and parasites, as well as in mitochondria.[2] Synthesis of saturated fatty acids via Fatty Acid Synthase II in E. coli

In animals, as well as yeast and some fungi, these same reactions occur on fatty acid synthase I (FASI), a large dimeric protein that has all of the enzymatic activities required to create a fatty acid. FASI is less efficient than FASII; however, it allows for the formation of more molecules, including “medium-chain” fatty acids via early chain termination.[2] Once a 16:0 carbon fatty acid has been formed, it can undergo a number of modifications, in particular by fatty acid synthase III (FASIII), which uses 2 carbon molecules to elongate preformed fatty acids.[2] Step

Enzyme

Reaction

Description

(a)

Acetyl CoA:ACP transacylase

Activates acetyl CoA for reaction with malonyl-ACP

(b)

Malonyl CoA:ACP transacylase

Activates malonyl CoA for reaction with acetyl-ACP

Fatty acid synthesis

260

(c)

3-ketoacyl-ACP synthetase

Reacts priming acetyl-ACP with chain-extending malonyl-ACP.

(d)

3-ketoacyl-ACP reductase

Reduces the carbon 3 ketone to a hydroxyl group

(e)

3-Hydroxyacyl ACP dehydrase

Removes water

(f)

Enoyl-ACP reductase

Reduces the C3-C4 double bond.

Abbreviations: ACP - Acyl carrier protein, CoA - Coenzyme A, NADP - Nicotinamide adenine dinucleotide phosphate. Regulation Acetyl-CoA is formed into malonyl-CoA by acetyl-CoA carboxylase, at which point malonyl-CoA is destined to feed into the fatty acid synthesis pathway. Acetyl-CoA carboxylase is the point of regulation in saturated straight-chain fatty acid synthesis, and is subject to both phosphorylation and allosteric regulation. Regulation by phosphorylation occurs mostly in mammals, while allosteric regulation occurs in most organisms. Allosteric control occurs as feedback inhibition by palmitoyl-CoA and activation by citrate. When there are high levels of palmitoyl-CoA, the final product of saturated fatty acid synthesis, it allosterically inactivates acetyl-CoA carboxylase to prevent a build-up of fatty acids in cells. Citrate acts to activate acetyl-CoA carboxylase under high levels, because high levels indicate that there is enough acetyl-CoA to feed into the Krebs cycle and produce energy.[3] De Novo Synthesis in Humans In humans, fatty acids are formed predominantly in the liver and lactating mammary glands, and, to a lesser extent, the adipose tissue. Most acetyl-CoA is formed from pyruvate by pyruvate dehydrogenase in the mitochondria. Acetyl-CoA produced in the mitochondria is condensed with oxaloacetate by citrate synthase to form citrate, which is then transported into the cytosol and broken down to yield acetyl-CoA and oxaloacetate by ATP citrate lyase. Oxaloacetate in the cytosol is reduced to malate by cytoplasmic malate dehydrogenase, and malate is transported back into the mitochondria to participate in the Citric acid cycle.[4]

Desaturation Desaturation of fatty acids involves a process that requires molecular oxygen (O2), NADH, and cytochrome b5. The reaction, which occurs in the endoplasmic reticulum, results in the oxidation of both the fatty acid and NADH. The most common desaturation reactions involve the placement of a double bond between carbons 9 and 10 (as in the conversion of palmitic acid to palmitoleic acid and the conversion of stearic acid to oleic acid, facilitated by the action of Δ9-desaturase). Other positions that can be desaturated in humans include carbon 4, 5, and 6, via Δ4-, Δ5-, and Δ6-desaturases, respectively.

Fatty acid synthesis Unsaturated fatty acids are essential components to prokaryotic and eukaryotic cell membranes. These fatty acids function primarily in maintaining membrane fluidity.[5] They have also been associated with serving as signaling molecules in other processes such as cell differentiation and DNA replication.[5] There are two pathways organisms use for desaturation: Aerobic and Anaerobic. Anaerobic Desaturation Many bacteria use the anaerobic pathway for synthesizing unsaturated fatty acids. This pathway does not utilize oxygen and is dependent on enzymes to insert the double bond before elongation utilizing the normal fatty acid synthesis machinery. In Escherichia coli, this pathway is well understood. • FabA is a β-hydroxydecanoyl-ACP dehydrase - it is specific for the 10-carbon saturated fatty acid synthesis intermediate (β-hydroxydecanoyl-ACP). • FabA catalyzes the dehydration of β-hydroxydecanoyl-ACP, causing the release of water and insertion of the double bond between C7 and C8 counting from the methyl end. This creates the trans-2-decenoyl intermediate. • Either the trans-2-decenoyl intermediate can be shunted to the normal saturated fatty acid synthesis pathway by FabB, where the double bond will be hydrolyzed and the final product will be a saturated fatty acid, or FabA will catalyze the isomerization into the Synthesis of unsaturated fatty acids via anaerobic cis-3-decenoyl intermediate. desaturation • FabB is a β-ketoacyl-ACP synthase that elongates and channels intermediates into the mainstream fatty acid synthesis pathway. When FabB reacts with the cis-decenoyl intermediate, the final product after elongation will be an unsaturated fatty acid.[6] • The two main unsaturated fatty acids made are Palmitoleoyl-ACP (16:1ω7) and cis-vaccenoyl-ACP (18:1ω7).[7] Most bacteria that undergo anaerobic desaturation contain homologues of FabA and FabB.[8] Clostridia are the main exception; they have a novel enzyme, yet to be identified, that catalyzes the formation of the cis double bond.[7] Regulation This pathway undergoes transcriptional regulation by FadR and FabR. FadR is the more extensively studied protein and has been attributed bifunctional characteristics. It acts as an activator of fabA and fabB transcription and as a repressor for the β-oxidation regulon. In contrast, FabR acts as a repressor for the transcription of fabA and fabB.[6]

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Fatty acid synthesis Aerobic Desaturation Aerobic desaturation is the most widespread pathway for the synthesis of unsaturated fatty acids. It is utilized in all eukaryotes and some prokaryotes. This pathway utilizes desaturases to synthesize unsaturated fatty acids from full-length saturated fatty acid substrates.[9] All desaturases require oxygen and ultimately consume NADH even though desaturation is an oxidative process. Desaturases are specific for the double bond they induce in the substrate. In Bacillus subtilis, the desaturase, Δ5-Des, is specific for inducing a cis-double bond at the Δ5 position.[5][9] Saccharomyces cerevisiae contains one desaturase, Ole1p, which induces the cis-double bond at Δ9.[5] Regulation In B. subtilis, this pathway is regulated by a two-component system: Synthesis of unsaturated fatty acids via aerobic DesK and DesR. DesK is a membrane-associated kinase and DesR is a desaturation transcriptional regulator of the des gene.[5][9] The regulation responds to temperature; when there is a drop in temperature, this gene is upregulated. Unsaturated fatty acids increase the fluidity of the membrane and stabilize it under lower temperatures. DesK is the sensor protein that, when there is a decrease in temperature, will autophosphorylate. DesK-P will transfer its phosphoryl group to DesR. Two DesR-P proteins will dimerize and bind to the DNA promoters of the des gene and recruit RNA polymerase to begin transcription.[5][9] Pseudomonas aeruginosa In general, both anaerobic and aerobic unsaturated fatty acid synthesis will not occur within the same system, however Pseudomonas aeruginosa and Vibrio ABE-1 are exceptions.[10][11][12] While, P. aeruginosa undergoes primarily anaerobic desaturation, it also undergoes two aerobic pathways. One pathway utilizes a Δ9-desaturase (DesA) that catalyzes a double bond formation in membrane lipids. Another pathway uses two proteins, DesC and DesB, together to act as a Δ9-desaturase, which inserts a double bond into a saturated fatty acid-CoA molecule. This second pathway is regulated by repressor protein DesT. DesT is also a repressor of fabAB expression for anaerobic desaturation when in presence of exogenous unsaturated fatty acids. This functions to coordinate the expression of the two pathways within the organism.[11][13]

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Branched-chain fatty acids Branched-chain fatty acids are usually saturated and are found in two distinct families: the iso-series and anteiso-series. It has been found that Actinomycetales contain unique branch-chain fatty acid synthesis mechanisms, including that which forms tuberculosteric acid.

Branch-Chain Fatty Acid Synthesizing System

Valine primer

Leucine primer

263

Fatty acid synthesis

Isoleucine primer Synthetic pathways of the branched-chain fatty acid synthesizing system given differing primers The branched-chain fatty acid synthesizing system uses α-keto acids as primers. This system is distinct from the branched-chain fatty acid synthetase that utilizes short-chain acyl-CoA esters as primers.[14] α-Keto acid primers are derived from the transamination and decarboxylation of valine, leucine, and isoleucine to form 2-methylpropanyl-CoA, 3-methylbutyryl-CoA, and 2-Methylbutyryl-CoA, respectively.[15] 2-Methylpropanyl-CoA primers derived from valine are elongated to produce even-numbered iso-series fatty acids such as 14-methyl-pentadecanoic (isopalmitic) acid, and 3-methylbutyryl-CoA primers from leucine may be used to form odd-numbered iso-series fatty acids such as 13-methyl-tetradecanoic acid. 2-Methylbutyryl-CoA primers from isoleucine are elongated to form anteiso-series fatty acids containing an odd number of carbon atoms such as 12-Methyl tetradecanoic acid.[16] Decarboxylation of the primer precursors occurs through the branched-chain α-keto acid decarboxylase (BCKA) enzyme. Elongation of the fatty acid follows the same biosynthetic pathway in Escherichia coli used to produce straight-chain fatty acids where malonyl-CoA is used as a chain extender.[17] The major end products are 12-17 carbon branched-chain fatty acids and their composition tends to be uniform and characteristic for many bacterial species.[16] BCKA decarboxylase and relative activities of α-keto acid substrates The BCKA decarboxylase enzyme is composed of two subunits in a tetrameric structure (A2B2) and is essential for the synthesis of branched-chain fatty acids. It is responsible for the decarboxylation of α-keto acids formed by the transamination of valine, leucine, and isoleucine and produces the primers used for branched-chain fatty acid synthesis. The activity of this enzyme is much higher with branched-chain α-keto acid substrates than with straight-chain substrates, and in Bacillus species its specificity is highest for the isoleucine-derived α-keto-β-methylvaleric acid, followed by α-ketoisocaproate and α-ketoisovalerate.[16][17] The enzyme’s high affinity toward branched-chain α-keto acids allows it to function as the primer donating system for branched-chain fatty acid synthetase.[17]

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Substrate

BCKA activity CO2 Produced (nmol/min mg) Km (μM) Vmax (nmol/min mg)

-α-keto-β-methyl-valerate 100%

19.7