Succinate dehydrogenase or succinate-coenzyme Q reductase or respiratory Complex II is an enzyme complex, found in many bacterial cells and in the inner mitochondrial membrane of eukaryotes. It is the only enzyme that participates in both the citric acid cycle and the electron transport chain. Histochemical analysis showing high succinate dehydrogenase in muscle demonstrates high mitochondrial content and high oxidative potential. In step 6 of the citric acid cycle, SQR catalyzes the oxidation of succinate to fumarate with the reduction of ubiquinone to ubiquinol; this occurs in the inner mitochondrial membrane by coupling the two reactions together. Mitochondrial and many bacterial monomer SQRs are composed of four subunits: two hydrophilic and two hydrophobic; the first two subunits, a flavoprotein and an iron-sulfur protein, are hydrophilic. SdhA contains a covalently attached flavin adenine dinucleotide cofactor and the succinate binding site and SdhB contains three iron-sulfur clusters:, and.
The second two subunits are hydrophobic membrane anchor subunits, SdhC and SdhD. Human mitochondria contain two distinct isoforms of SdhA, these isoforms are found in Ascaris suum and Caenorhabditis elegans; the subunits form a membrane-bound cytochrome b complex with six transmembrane helices containing one heme b group and a ubiquinone-binding site. Two phospholipid molecules, one cardiolipin and one phosphatidylethanolamine, are found in the SdhC and SdhD subunits, they serve to occupy the hydrophobic space below the heme b. These subunits are displayed in the attached image. SdhA is green, SdhB is teal, SdhC is fuchsia, SdhD is yellow. Around SdhC and SdhD is a phospholipid membrane with the intermembrane space at the top of the image. Ubiquinone's binding site, image 4, is located in a gap composed of SdhB, SdhC, SdhD. Ubiquinone is stabilized by the side chains of His207 of subunit B, Ser27 and Arg31 of subunit C, Tyr83 of subunit D; the quinone ring is surrounded by Ile28 of subunit C and Pro160 of subunit B.
These residues, along with Il209, Trp163, Trp164 of subunit B, Ser27 of subunit C, form the hydrophobic environment of the quinone-binding pocket. SdhA provides the binding site for the oxidation of succinate; the side chains Thr254, His354, Arg399 of subunit A stabilize the molecule while FAD oxidizes and carries the electrons to the first of the iron-sulfur clusters. This can be seen in image 5; the succinate-binding site and ubiquinone-binding site are connected by a chain of redox centers including FAD and the iron-sulfur clusters. This chain extends over 40 Å through the enzyme monomer. All edge-to-edge distances between the centers are less than the suggested 14 Å limit for physiological electron transfer; this electron transfer is demonstrated in image 8. Little is known about the exact succinate oxidation mechanism. However, the crystal structure shows that FAD, Glu255, Arg286, His242 of subunit A are good candidates for the initial deprotonation step. Thereafter, there are two possible elimination mechanisms: E1cb.
In the E2 elimination, the mechanism is concerted. The basic residue or cofactor deprotonates the alpha carbon, FAD accepts the hydride from the beta carbon, oxidizing the bound succinate to fumarate—refer to image 6. In E1cb, an enolate intermediate is shown in image 7, before FAD accepts the hydride. Further research is required to determine which elimination mechanism succinate undergoes in Succinate Dehydrogenase. Oxidized fumarate, now loosely bound to the active site, is free to exit the protein. After the electrons are derived from succinate oxidation via FAD, they tunnel along the relay until they reach the cluster; these electrons are subsequently transferred to an awaiting ubiquinone molecule within the active site. The Iron-Sulfur electron tunneling system is shown in image 9; the O1 carbonyl oxygen of ubiquinone is oriented at the active site by hydrogen bond interactions with Tyr83 of subunit D. The presence of electrons in the iron sulphur cluster induces the movement of ubiquinone into a second orientation.
This facilitates a second hydrogen bond interaction between the O4 carbonyl group of ubiquinone and Ser27 of subunit C. Following the first single electron reduction step, a semiquinone radical species is formed; the second electron arrives from the cluster to provide full reduction of the ubiquinone to ubiquinol. This mechanism of the ubiquinone reduction is shown in image 8. Although the functionality of the heme in succinate dehydrogenase is still being researched, some studies have asserted that the first electron delivered to ubiquinone via may tunnel back and forth between the heme and the ubiquinone intermediate. In this way, the heme cofactor acts as an electron sink, its role is to prevent the interaction of the intermediate with molecular oxygen to produce reactive oxygen species. The heme group, relative to ubiquinone, is shown in image 4, it has been proposed that a gating mechanism may be in place to prevent the electrons from tunneling directly to the heme from the cluster. A potential candidate is residue His207, which lies directly between the heme.
His207 of subunit B is in direct proximity to the cluster, the bound ubiquinone, the heme. To reduce the quinone in SQR, two electrons as well as two protons are needed, it has been argued that a water molecule arrives at the active site and is coordinated by His207 of subunit B, Arg31 of subunit C, Asp82 of subunit D. The semiquinone species is protonated by protons deliv
Peroxidases are a large family of enzymes which play a role in various biological processes. Peroxidases catalyze a reaction of the form: ROOR ′ + 2 e − electron donor + 2 H + → Peroxidase ROH + R ′ OH For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides. Peroxidases can contain a heme cofactor in their active sites, or alternately redox-active cysteine or selenocysteine residues; the nature of the electron donor is dependent on the structure of the enzyme. For example, horseradish peroxidase can use a variety of organic compounds as electron donors and acceptors. Horseradish peroxidase has an accessible active site, many compounds can reach the site of the reaction. On the other hand, for an enzyme such as cytochrome c peroxidase, the compounds that donate electrons are specific, due to a narrow active site; the glutathione peroxidase family consists of 8 known human isoforms. Glutathione peroxidases use glutathione as an electron donor and are active with both hydrogen peroxide and organic hydroperoxide substrates.
Gpx1, Gpx2, Gpx3, Gpx4 have been shown to be selenium-containing enzymes, whereas Gpx6 is a selenoprotein in humans with cysteine-containing homologues in rodents. Amyloid beta, when bound to heme, has been shown to have peroxidase activity. A typical group of peroxidases are the haloperoxidases; this group is able to form reactive halogen species and, as a result, natural organohalogen substances. A majority of peroxidase protein sequences can be found in the PeroxiBase database. While the exact mechanisms have yet to be determined, peroxidases are known to play a part in increasing a plant's defenses against pathogens. Many members of the Solanaceae, notably Solanum melongena and Capsicum chinense use Guaiacol and the enzyme guaiacol peroxidase as a defense against the bacterial parasite Ralstonia solanacearum: the gene expression for this enzyme commences within minutes of bacterial attack. Peroxidase can be used for treatment of industrial waste waters. For example, which are important pollutants, can be removed by enzyme-catalyzed polymerization using horseradish peroxidase.
Thus phenols are oxidized to phenoxy radicals, which participate in reactions where polymers and oligomers are produced that are less toxic than phenols. It can be used to convert toxic materials into less harmful substances. There are many investigations about the use of peroxidase in many manufacturing processes like adhesives, computer chips, car parts, linings of drums and cans. Other studies have shown that peroxidases may be used to polymerize anilines and phenols in organic solvent matrices. Peroxidases are sometimes used as histological markers. Cytochrome c peroxidase is used as a soluble purified model for cytochrome c oxidase. Animal heme-dependent peroxidases Ascorbate peroxidase Catalase Chloride peroxidase Cytochrome c peroxidase Di-haem cytochrome c peroxidase Glutathione peroxidase Haloperoxidase Hemoprotein Lactoperoxidase Manganese peroxidase Myeloperoxidase Peroxide Peroxiredoxin Thyroid peroxidase Vanadium bromoperoxidase
Pentose phosphate pathway
The pentose phosphate pathway is a metabolic pathway parallel to glycolysis. It generates NADPH and pentoses as well as ribose 5-phosphate, the last one a precursor for the synthesis of nucleotides. While it does involve oxidation of glucose, its primary role is anabolic rather than catabolic; the pathway is important in red blood cells. There are two distinct phases in the pathway; the first is the oxidative phase, in which NADPH is generated, the second is the non-oxidative synthesis of 5-carbon sugars. For most organisms, the pentose phosphate pathway takes place in the cytosol. Similar to glycolysis, the pentose phosphate pathway appears to have a ancient evolutionary origin; the reactions of this pathway are enzyme-catalyzed in modern cells, they occur non-enzymatically under conditions that replicate those of the Archean ocean, are catalyzed by metal ions ferrous ions. This suggests; the primary results of the pathway are: The generation of reducing equivalents, in the form of NADPH, used in reductive biosynthesis reactions within cells.
Production of ribose 5-phosphate, used in the synthesis of nucleic acids. Production of erythrose 4-phosphate used in the synthesis of aromatic amino acids. Aromatic amino acids, in turn, are precursors for many biosynthetic pathways, including the lignin in wood. Dietary pentose sugars derived from the digestion of nucleic acids may be metabolized through the pentose phosphate pathway, the carbon skeletons of dietary carbohydrates may be converted into glycolytic/gluconeogenic intermediates. In mammals, the PPP occurs in the cytoplasm, 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 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 generated for phagocytes in a process referred to as a respiratory burst. 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; the entire set of reactions can be summarized as follows: The overall reaction for this process is: Glucose 6-phosphate + 2 NADP+ + H2O → ribulose 5-phosphate + 2 NADPH + 2 H+ + CO2 Net reaction: 3 ribulose-5-phosphate → 1 ribose-5-phosphate + 2 xylulose-5-phosphate → 2 fructose-6-phosphate + glyceraldehyde-3-phosphate Glucose-6-phosphate dehydrogenase is the rate-controlling enzyme of this pathway. It is allosterically stimulated by NADP+ and inhibited by NADPH; the ratio of NADPH:NADP+ is 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 inhibited by acetyl CoA. G6PD activity is post-translationally regulated by cytoplasmic deacetylase SIRT2. SIRT2-mediated deacetylation and activation of G6PD stimulates oxidative branch of PPP to supply cytosolic NADPH to counteract oxidative damage or support de novo lipogenesis. Several deficiencies in the level of activity 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 such that it cannot sustain the parasitic life cycle long enough for productive growth. G6PD deficiency – A hereditary disease that disrupts the pentose phosphate pathway RNA Thiamine deficiency Frank Dickens FRS The chemical logic behind the pentose phosphate pathway Pentose+Phosphate+Pathway at the US National Library of Medicine Medical Subject Headings Pentose phosphate pathway Map – Homo sapiens
Acetaldehyde dehydrogenases are dehydrogenase enzymes which catalyze the conversion of acetaldehyde into acetic acid. The oxidation of acetaldehyde to acetate can be summarized as follows: Acetaldehyde + NAD+ + Coenzyme A ↔ Acetyl-CoA + NADH + H+In humans, there are three known genes which encode this enzymatic activity, ALDH1A1, ALDH2, the more discovered ALDH1B1; these enzymes are members of the larger class of aldehyde dehydrogenases. The CAS number for this type of the enzyme is. Cysteine-302 is one of three consecutive Cys residues and is crucial to the enzyme’s catalytic function; the residue is alkylated by iodoacetamide in both the cytosolic and mitochondrial isozymes, with modifications to Cys-302 indicative of catalytic activity with other residues. Furthermore, the preceding sequence Gln-Gly-Gln-Cys is conserved in both isozymes for both human and horse, consistent with Cys-302 being crucial to catalytic function; as discovered by site-directed mutagenesis, glutamate-268 is a key component of liver acetaldehyde dehydrogenase and is critical to catalytic activity.
Since activity in mutants could not be restored by addition of general bases, it’s suggested that the residue functions as a general base for activation of the essential Cys-302 residue. In bacteria, acylating acetaldehyde dehydrogenase forms a bifunctional heterodimer with metal-dependent 4-hydroxy-2-ketovalerate aldolase. Utilized in the bacterial degradation of toxic aromatic compounds, the enzyme’s crystal structure indicates that intermediates are shuttled directly between active sites through a hydrophobic intermediary channel, providing an unreactive environment in which to move the reactive acetaldehyde intermediate from the aldolase active site to the acetaldehyde dehydrogenase active site; such communication between proteins allows for the efficient transfer substrates from one active site to the next. Although the two isozymes do not share a common subunit, the homology between the human ALDH1 and ALDH2 proteins is 66% at the coding nucleotide level and 69% at the amino acid level, found to be lower than the 91% homology between human ALDH1 and horse ALDH1.
Such a finding is consistent with evidence suggesting the early evolutionary divergence between cytosolic and mitochondrial isozymes, as seen in the 50% homology between pig mitochondrial and cytosolic asparatate aminotransferases. In the liver, ethanol is converted into the harmless acetic acid by acetaldehyde dehydrogenase. Acetaldehyde is responsible for many hangover symptoms. About 50% of people of Northeast Asian descent have a dominant mutation in their acetaldehyde dehydrogenase gene, making this enzyme less effective. A similar mutation is found in about 5–10% of blond-haired blue-eyed people of Northern European descent. In these people, acetaldehyde accumulates after drinking alcohol, leading to symptoms of acetaldehyde poisoning, including the characteristic flushing of the skin and increased heart and respiration rates. Other symptoms can include severe abdominal and urinary tract cramping and cold flashes, profuse sweating, profound malaise. Individuals with deficient acetaldehyde dehydrogenase activity are far less to become alcoholics, but seem to be at a greater risk of liver damage, alcohol-induced asthma, contracting cancers of the oro-pharynx and esophagus due to acetaldehyde overexposure.
This demonstrates that many of ethanol's toxic effects are mediated via the acetaldehyde metabolite and can therefore be mitigated by substances such as fomepizole which reduces the conversion rate of ethanol to acetaldehyde in vivo. ALDH2, which has a lower KM for acetaldehydes than ALDH1 and acts predominantly in the mitochondrial matrix, is the main enzyme in acetaldehyde metabolism and has three genotypes. A single point mutation at exon 12 of the ALDH2 gene causes a replacement of glutamine with lysine at residue 487, resulting in the ALDH2K enzyme. ALDH2K has an increased KM for NAD+, rendering it inactive at cellular concentrations of NAD+. Since ALDH2 is a randomized tetramer, the hetero-mutated genotype is reduced to only 6% activity compared to wild type, while homo-mutated genotypes have zero enzyme activity; the ALDH2-deficient subunit is dominant in hybridization with a wild type subunit, resulting in inactivation of the isozyme by interfering with catalytic activity and increasing turnover.
ALDH2 genetic variation has been correlated with alcohol dependence, with heterozygotes at a reduced risk compared to wild type homozygotes and individual homozygotes for the ALDH2-deficient at a low risk for alcoholism. The drug disulfiram prevents the oxidation of acetaldehyde to acetic acid and is used in the treatment of alcoholism. ALDH1 is inhibited by disulfiram, while ALDH2 is resistant to its effect; the cysteine residue at 302 in ALDH1 and 200 in ALDH2 is implicated as a disulfiram binding site on the enzyme and serves as a disfulfiram sensitive thiol site. Covalent binding of disulfiram to the thiol blocks the binding of one of the cysteine residues with iodoacetamide, thereby inactivating the enzyme and lowering catalytic activity. Activity can be recovered by treatment with 2-mercaptoethanol, although not with glutathione. Metronidazole, used to treat certain parasitic infections as well as pseudomembranous colitis, causes similar effects to disulfiram. Coprine metabolizes in vivo to 1-aminocyclopropanol.
ALDH1 is involved in the metabolism of Vitamin A. Animal models suggest that absence of the gene is associated with protection against visceral adiposity
Nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide is a cofactor found in all living cells. The compound is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains the other nicotinamide. Nicotinamide adenine dinucleotide exists in two forms: an oxidized and reduced form, abbreviated as NAD+ and NADH respectively. In metabolism, nicotinamide adenine dinucleotide is involved in redox reactions, carrying electrons from one reaction to another; the cofactor is, found in two forms in cells: NAD+ is an oxidizing agent – it accepts electrons from other molecules and becomes reduced. This reaction forms NADH, which can be used as a reducing agent to donate electrons; these electron transfer reactions are the main function of NAD. However, it is used in other cellular processes, most notably a substrate of enzymes that add or remove chemical groups from proteins, in posttranslational modifications; because of the importance of these functions, the enzymes involved in NAD metabolism are targets for drug discovery.
In organisms, NAD can be synthesized from simple building-blocks from the amino acids tryptophan or aspartic acid. In an alternative fashion, more complex components of the coenzymes are taken up from food as niacin. Similar compounds are released by reactions that break down the structure of NAD; these preformed components pass through a salvage pathway that recycles them back into the active form. Some NAD is converted into the coenzyme nicotinamide adenine dinucleotide phosphate; the chemistry of NADP is similar to that of NAD, but it has different role, being predominantly a cofactor in anabolic metabolism. NAD+ is written with a superscript plus sign because of the formal charge on one of its nitrogen atoms. NADH, on the other hand, is a doubly charged anion because of its two bridging phosphate groups. Nicotinamide adenine dinucleotide, like all dinucleotides, consists of two nucleosides joined by a pair of bridging phosphate groups; the nucleosides each contain a ribose ring, one with adenine attached to the first carbon atom and the other with nicotinamide at this position.
The nicotinamide moiety can be attached in two orientations to this anomeric carbon atom. Because of these two possible structures, the compound exists as two diastereomers, it is the β-nicotinamide diastereomer of NAD+, found in organisms. These nucleotides are joined together by a bridge of two phosphate groups through the 5' carbons. In metabolism, the compound donates electrons in redox reactions; such reactions involve the removal of two hydrogen atoms from the reactant, in the form of a hydride ion, a proton. The proton is released into solution, while the reductant RH2 is oxidized and NAD+ reduced to NADH by transfer of the hydride to the nicotinamide ring. RH2 + NAD+ → NADH + H+ + R; the midpoint potential of the NAD+/NADH redox pair is −0.32 volts, which makes NADH a strong reducing agent. The reaction is reversible, when NADH reduces another molecule and is re-oxidized to NAD+; this means the coenzyme can continuously cycle between the NAD+ and NADH forms without being consumed. In appearance, all forms of this coenzyme are white amorphous powders that are hygroscopic and water-soluble.
The solids are stable. Solutions of NAD+ are colorless and stable for about a week at 4 °C and neutral pH, but decompose in acids or alkalis. Upon decomposition, they form products. Both NAD+ and NADH absorb ultraviolet light because of the adenine. For example, peak absorption of NAD+ is at a wavelength of 259 nanometers, with an extinction coefficient of 16,900 M−1cm−1. NADH absorbs at higher wavelengths, with a second peak in UV absorption at 339 nm with an extinction coefficient of 6,220 M−1cm−1; this difference in the ultraviolet absorption spectra between the oxidized and reduced forms of the coenzymes at higher wavelengths makes it simple to measure the conversion of one to another in enzyme assays – by measuring the amount of UV absorption at 340 nm using a spectrophotometer. NAD+ and NADH differ in their fluorescence. NADH in solution has an emission peak at 460 nm and a fluorescence lifetime of 0.4 nanoseconds, while the oxidized form of the coenzyme does not fluoresce. The properties of the fluorescence signal changes when NADH binds to proteins, so these changes can be used to measure dissociation constants, which are useful in the study of enzyme kinetics.
These changes in fluorescence are used to measure changes in the redox state of living cells, through fluorescence microscopy. In rat liver, the total amount of NAD+ and NADH is 1 μmole per gram of wet weight, about 10 times the concentration of NADP+ and NADPH in the same cells; the actual concentration of NAD+ in cell cytosol is harder to measure, with recent estimates in animal cells ranging around 0.3 mM, 1.0 to 2.0 mM in yeast. However, more than 80% of NADH fluorescence in mitochondria is from bound form, so the concentration in solution is much lower. Data for other compartments in the cell are limited, although in the mitochondrion the concentration of NAD+ is similar to that in the cytosol; this NAD+ is carried into the mitochondrion by a specific membrane transport protein, since the coenzyme cannot diffuse across membranes. The balance between the oxidized and reduced forms of nicotinam
A cofactor is a non-protein chemical compound or metallic ion, required for an enzyme's activity. Cofactors can be considered "helper molecules"; the rates at which these happen are characterized by enzyme kinetics. Cofactors can be subclassified as either inorganic ions or complex organic molecules called coenzymes, the latter of, derived from vitamins and other organic essential nutrients in small amounts. A coenzyme, or covalently bound is termed a prosthetic group. Cosubstrates are transiently bound to the protein and will be released at some point get back in; the prosthetic groups, on the other hand, are bound permanently to the protein. Both of them have the same function, to facilitate the reaction of enzymes and protein. Additionally, some sources limit the use of the term "cofactor" to inorganic substances. An inactive enzyme without the cofactor is called an apoenzyme, while the complete enzyme with cofactor is called a holoenzyme; some enzymes or enzyme complexes require several cofactors.
For example, the multienzyme complex pyruvate dehydrogenase at the junction of glycolysis and the citric acid cycle requires five organic cofactors and one metal ion: loosely bound thiamine pyrophosphate, covalently bound lipoamide and flavin adenine dinucleotide, cosubstrates nicotinamide adenine dinucleotide and coenzyme A, a metal ion. Organic cofactors are vitamins or made from vitamins. Many contain the nucleotide adenosine monophosphate as part of their structures, such as ATP, coenzyme A, FAD, NAD+; this common structure may reflect a common evolutionary origin as part of ribozymes in an ancient RNA world. It has been suggested that the AMP part of the molecule can be considered to be a kind of "handle" by which the enzyme can "grasp" the coenzyme to switch it between different catalytic centers. Cofactors can be divided into two major groups: organic Cofactors, such as flavin or heme, inorganic cofactors, such as the metal ions Mg2+, Cu+, Mn2+, or iron-sulfur clusters. Organic cofactors are sometimes further divided into prosthetic groups.
The term coenzyme refers to enzymes and, as such, to the functional properties of a protein. On the other hand, "prosthetic group" emphasizes the nature of the binding of a cofactor to a protein and, refers to a structural property. Different sources give different definitions of coenzymes and prosthetic groups; some consider bound organic molecules as prosthetic groups and not as coenzymes, while others define all non-protein organic molecules needed for enzyme activity as coenzymes, classify those that are bound as coenzyme prosthetic groups. These terms are used loosely. A 1980 letter in Trends in Biochemistry Sciences noted the confusion in the literature and the arbitrary distinction made between prosthetic groups and coenzymes group and proposed the following scheme. Here, cofactors were defined as an additional substance apart from protein and substrate, required for enzyme activity and a prosthetic group as a substance that undergoes its whole catalytic cycle attached to a single enzyme molecule.
However, the author could not arrive at a single all-encompassing definition of a "coenzyme" and proposed that this term be dropped from use in the literature. Metal ions are common cofactors; the study of these cofactors falls under the area of bioinorganic chemistry. In nutrition, the list of essential trace elements reflects their role as cofactors. In humans this list includes iron, manganese, copper and molybdenum. Although chromium deficiency causes impaired glucose tolerance, no human enzyme that uses this metal as a cofactor has been identified. Iodine is an essential trace element, but this element is used as part of the structure of thyroid hormones rather than as an enzyme cofactor. Calcium is another special case, in that it is required as a component of the human diet, it is needed for the full activity of many enzymes, such as nitric oxide synthase, protein phosphatases, adenylate kinase, but calcium activates these enzymes in allosteric regulation binding to these enzymes in a complex with calmodulin.
Calcium is, therefore, a cell signaling molecule, not considered a cofactor of the enzymes it regulates. Other organisms require additional metals as enzyme cofactors, such as vanadium in the nitrogenase of the nitrogen-fixing bacteria of the genus Azotobacter, tungsten in the aldehyde ferredoxin oxidoreductase of the thermophilic archaean Pyrococcus furiosus, cadmium in the carbonic anhydrase from the marine diatom Thalassiosira weissflogii. In many cases, the cofactor includes both an organic component. One diverse set of examples is the heme proteins, which consist of a porphyrin ring coordinated to iron. Iron-sulfur clusters are complexes of iron and sulfur atoms held within proteins by cysteinyl residues, they play both structural and functional roles, including electron transfer, redox sensing, as structural modules. Organic cofactors are small organic molecules that can be either loosely or bound to the enzyme and directly participate in the reaction. In the latter case, when it is difficult to remove without denaturing the enzyme, it can be called a prosthetic group.
It is important to emphasize that there is no sharp division between loosely and bound cofactors. Indeed, many such as NAD+ can be bound in some enzymes, while it is loosely bound in others. Another example is thiamine pyrophosphate, bound in transketolase or pyruvate decarboxylase, while it is less tightly
Glutamic acid is an α-amino acid, used by all living beings in the biosynthesis of proteins. It is non-essential in humans, it is an excitatory neurotransmitter, in fact the most abundant one, in the vertebrate nervous system. It serves as the precursor for the synthesis of the inhibitory gamma-aminobutyric acid in GABA-ergic neurons, it has a formula C5H9O4N. Its molecular structure could be idealized as HOOC-CH-2-COOH, with two carboxyl groups -COOH and one amino group -NH2. However, in the solid state and mildly acid water solutions, the molecule assumes an electrically neutral zwitterion structure −OOC-CH-2-COOH, it is encoded by the codons GAA or GAG. The acid can lose one proton from its second carboxyl group to form the conjugate base, the singly-negative anion glutamate −OOC-CH-2-COO−; this form of the compound is prevalent in neutral solutions. The glutamate neurotransmitter plays the principal role in neural activation; this anion is responsible for the savory flavor of certain foods, used in glutamate flavorings such as MSG.
In Europe it is classified as food additive E620. In alkaline solutions the doubly negative anion −OOC-CH-2-COO− prevails; the radical corresponding to glutamate is called glutamyl. When glutamic acid is dissolved in water, the amino group may gain a proton, and/or the carboxyl groups may lose protons, depending on the acidity of the medium. In sufficiently acidic environments, the amino group gains a proton and the molecule becomes a cation with a single positive charge, HOOC-CH-2-COOH. At pH values between about 2.5 and 4.1, the carboxylic acid closer to the amine loses a proton, the acid becomes the neutral zwitterion −OOC-CH-2-COOH. This is the form of the compound in the crystalline solid state; the change in protonation state is gradual. At higher pH, the other carboxylic acid group loses its proton and the acid exists entirely as the glutamate anion −OOC-CH-2-COO−, with a single negative charge overall; the change in protonation state occurs at pH 4.07. This form with both carboxylates lacking protons is dominant in the physiological pH range.
At higher pH, the amino group loses the extra proton and the prevalent species is the doubly-negative anion −OOC-CH-2-COO−. The change in protonation state occurs at pH 9.47. The carbon atom adjacent to the amino group is chiral, so glutamic acid can exist in two optical isomers, D and L; the L form is the one most occurring in nature, but the D form occurs in some special contexts, such as the cell walls of the bacteria and the liver of mammals. Although they occur in many foods, the flavor contributions made by glutamic acid and other amino acids were only scientifically identified early in the twentieth century; the substance was discovered and identified in the year 1866, by the German chemist Karl Heinrich Ritthausen who treated wheat gluten with sulfuric acid. In 1908 Japanese researcher Kikunae Ikeda of the Tokyo Imperial University identified brown crystals left behind after the evaporation of a large amount of kombu broth as glutamic acid; these crystals, when tasted, reproduced the ineffable but undeniable flavor he detected in many foods, most in seaweed.
Professor Ikeda termed this flavor umami. He patented a method of mass-producing a crystalline salt of glutamic acid, monosodium glutamate. Glutamic acid is produced on the largest scale of any amino acid, with an estimated annual production of about 1.5 million tons in 2006. Chemical synthesis was supplanted by the aerobic fermentation of sugars and ammonia in the 1950s, with the organism Corynebacterium glutamicum being the most used for production. Isolation and purification can be achieved by crystallization. Glutamate is a key compound in cellular metabolism. In humans, dietary proteins are broken down by digestion into amino acids, which serve as metabolic fuel for other functional roles in the body. A key process in amino acid degradation is transamination, in which the amino group of an amino acid is transferred to an α-ketoacid catalysed by a transaminase; the reaction can be generalised as such: R1-amino acid + R2-α-ketoacid ⇌ R1-α-ketoacid + R2-amino acidA common α-keto acid is α-ketoglutarate, an intermediate in the citric acid cycle.
Transamination of α-ketoglutarate gives glutamate. The resulting α-ketoacid product is a useful one as well, which can contribute as fuel or as a substrate for further metabolism processes. Examples are as follows: Alanine + α-ketoglutarate ⇌ pyruvate + glutamateAspartate + α-ketoglutarate ⇌ oxaloacetate + glutamateBoth pyruvate and oxaloacetate are key components of cellular metabolism, contributing as substrates or intermediates in fundamental processes such as glycolysis and the citric acid cycle. Glutamate plays an important role in the body's disposal of excess or waste nitrogen. Glutamate undergoes deamination, an oxidative reaction catalysed by glutamate dehydrogenase, as follows: glutamate + H2O + NADP+ → α-ketoglutarate + NADPH + NH3 + H+Ammonia is excreted predominantly as urea, synthesised in the liver. Transamination can thus be linked to deamination allowing nitrogen from the amine groups of amino acids to be removed, via glutamate as an intermediate, excreted from the body in the form of urea.
Glutamate is a