Aminotransferases pyridoxal-phosphate -dependent

FIGURE 14.22 Glutamate aspartate aminotransferase, an enzyme conforming to a double-displacement bisnbstrate mechanism. Glutamate aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme. The pyridoxal serves as the —NH, acceptor from glntamate to form pyridoxamine. Pyridoxamine is then the amino donor to oxaloacetate to form asparate and regenerate the pyridoxal coenzyme form. (The pyridoxamine enzyme is the E form.)... 

In addition to a-allenic a-amino acids, the corresponding allenic derivatives of y-aminobutyric acid (GABA) have also been synthesized as potential inhibitors of the pyridoxal phosphate-dependent enzyme GABA-aminotransferase (Scheme 18.49) [131,138-142]. The synthesis of y-allenyl-GABA (152) and its methylated derivatives was accomplished through Crabbe reaction [131], aza-Cope rearrangement [138] and lactam allenylation [139], whereas the fluoroallene 153 was prepared by SN2 -reduc-tion of a propargylic chloride [141]. 

This enzyme [EC 2.6.1.2], also known as glutamic-pyruvic transaminase and glutamic-alanine transaminase, catalyzes the pyridoxal-phosphate-dependent reaction of alanine with 2-ketoglutarate, resulting on the production of pyruvate and glutamate. 2-Aminobutanoate will also react, albeit slowly. There is another alanine aminotransferase [EC 2.6.1.12], better known as alanine-oxo-acid aminotransferase, which catalyzes the pyridoxal-phosphate-dependent reaction of alanine and a 2-keto acid to generate pyruvate and an amino acid. See also Alanine Glyoxylate Aminotransferase... 

This enzyme [EC 2.6.1.21], also known as D-aspartate aminotransferase, D-amino acid aminotransferase, and D-amino acid transaminase, catalyzes the reversible pyridoxal-phosphate-dependent reaction of D-alanine with a-ketoglutarate to yield pyruvate and D-glutamate. The enzyme will also utilize as substrates the D-stereoisomers of leucine, aspartate, glutamate, aminobutyrate, norva-hne, and asparagine. See o-Amino Acid Aminotransferase... 

This enzyme [EC 2.6.1.18], also known as j8-alanine-pyruvate aminotransferase, catalyzes the reversible pyridoxal-phosphate-dependent reaction of /3-alanine with pyruvate to generate 3-oxopropanoate and alanine. 

This enzyme [EC 2.6.1.42], also referred to as transaminase B, catalyzes the reversible reaction of leucine with a-ketoglutarate (or, 2-oxoglutarate) to produce 4-methyl-2-oxopentanoate and glutamate. The pyridoxal-phosphate-dependent enzyme will also utilize isoleucine and valine as substrates. However, this enzyme is distinct from that of valine pyruvate aminotransferase [EC 2.6.1.66]. See also Leucine Aminotransferase... 

This pyridoxal-phosphate-dependent enzyme [EC 2.6.1.4] catalyzes the reaction of glycine with a-ketoglu-tarate (or, 2-oxoglutarate) to produce glyoxylate and l-glutamate. See also GlycineiOxaloacetate Aminotransferase Glyoxylate Aminotransferase A. E. Braunstein (1973) The Enzymes, 3rd ed., 9, 379. 

Pyridoxal-phosphate-dependent enzymes, NXACETYLORNITHINE AMINOTRANSFERASE... 

Some pyridoxal phosphate-dependent enzymes are normally fuUy saturated with cofactor and show the same activity on assay in vitro whether additional pyridoxal phosphate is present in the incubation medium or not. Examples of this class of enzymes include liver cysteine sulfinate decarboxylase (which is involved in the synthesis of taurine from cysteine Section 14.5.1) and the brain and liver glutamate and aspartate aminotransferases. 

In all cases the keto acids seem to be formed by typical a-ketogluta-rate-linked, pyridoxal phosphate-dependent transaminases (EC 2.6.1.6, etc.) (9, 154, 156, 157). There has been little study of isolated, presumably specific enzymes in connection with flavors, although the leucine and alanine aminotransferases of tomato have been precipitated with (NH4)oS04 (164, 165). Transaminase activity in Saccharomyces cere-visiae has a pH optimum of 7.2 (154), and a-ketoglutarate is the only amino group recipient (154, 166). Only aspartate and amino acids with hydrophobic side chains are acted on (154). 

Historically, the amine was an aromatic amine but is now generalized to any amine. A Schiff base, also called an aldimine, is formed in the pyridoxal 5-phosphate-dependent aminotransferase reactions. 

L-Canaline is an ineffective antimetabolite of L-ornithine since it has little ability to antagonize ornithine-dependent reactions. On the other hand, it forms a covalently bound Schiff-base complex with the pyridoxal phosphate moiety of Bg-containing enzymes. As such it is a potent inhibitor of many decarboxylases and aminotransferases that utilize this vitamin. 

Aspartate aminotransferase is the prototype of a large family of PLP-dependent enzymes. Comparisons of amino acid sequences as well as several three-dimensional structures reveal that almost all transaminases having roles in amino acid biosynthesis are related to aspartate aminotransferase by divergent evolution. An examination of the aligned amino acid sequences reveals that two residues are completely conserved. These residues are the lysine residue that forms the Schiff base with the pyridoxal phosphate cofactor (lysine 258 in aspartate aminotransferase) and an arginine residue that interacts with the a-carboxylate group of the ketoacid (see Figure 23.11). 

Figure 23.12 Aspartate aminotransferase. The active site of this prototypical PLP-dependent enzyme includes pyridoxal phosphate attached to the enzyme by a Schiff-base linkage with lysine 2S8, An arginine residue in the active site helps orient substrates by binding to their u-carboxylate groups. Only one of the enzyme s two subunits is shown,...

The first step in the catabolism of most amino acids is the transfer of the o-amino group from the amino acid to a-ketoglutarate (tx-KG). This process is catalyzed by transaminase (aminotransferase) enzymes that require pyridoxal phosphate as a cofactor. The products of this reaction are glutamate (Glu) and the a-ketoacid analog of the amino acid destined for catabolic breakdown. For example, aspartate is converted to its a-keto analog, oxalo-acetate, by the action of aspartate transaminase (AST), which also produces Glu from a-KG. The transamination process is freely reversible, and the direction in which the reaction proceeds is dependent on the concentrations of the reactants and products. These reactions do not effect a net removal of amino nitrogen the amino group is only transferred from one amino acid to another. 

Isomerases and pyridoxal phosphate (PLP)-dependent transaminases (aminotransferases). The proton transfer reactions associated with isomerases and PLP-dependent transaminases are generally suprafacial processes. This may reflect a mechanistic advantage of a single active site base functioning in both proton abstraction and readdition at the same diastereotopic face of enediol(ate), dienol(ate), or enamine intermediates. 

Figure 1-2 ALA synthesis by ALAS or the C5 pathway. The enzymes of the C5 pathway and their cofactors are as follows (a) glutamyl-tRNA reductase (requires Mg-ATP) (b) glutamyl-tRNA reductase (pyridoxal 5 -phosphate dependent and requires NAD(P)H) and (c) glutamate 1-semialdehyde aminotransferase.

Fig. 38.14. The ornithine aminotransferase reaction. This is a reversible reaction dependent on pyridoxal phosphate, which normally favors ornithine degradation.

Hanford and Davies (1958) showed that a partly purilied extract from pea epicotyls converted phosphoglycerate to phosphoserine. The reaction was dependent on NAD, glutamate, and pyridoxal phosphate. The first step was presumably catalyzed by phosphoglycerate dehydrogenase and the second by glutamate phosphohydroxypyruvate aminotransferase. Such a phosphoserine aminotransferase was present in extracts of pea seeds, leaves, and apical meristems (Cheung et al., 1968). Little is known of phosphoserine phosphatase in plants, but its presence in spinach leaves is reported by Larsson and Albertsson (1979). 

Fig. 20.3 Pathway of methionine metabolism. The numbers represent the following enzymes or sequences (1) methionine adenosyltransferase (2) S-adenosylmethionine-dependent transmethylation reactions (3) glycine methyltransferase (4) S-adenosylhomocysteine hydrolase (5) betaine-homocysteine methyltransferase (6) 5-methyltetrahydrofolate homocysteine methyltransferase (7) serine hydroxymethyltransferase (8) 5,10-methylenetetrahydrofolate reductase (9) S-adenosylmethionine decarboxylase (10) spermidine and spermine synthases (11) methylthio-adenosine phosphorylase (12) conversion of methylthioribose to methionine (13) cystathionine P-synthase (14) cystathionine y-lyase (15) cysteine dioxygenase (16) cysteine suplhinate decarboxylase (17) hypotaurine NAD oxidoreductase (18) cysteine sulphintite a-oxoglutarate aminotransferase (19) sulfine oxidase. MeCbl = methylcobalamin PLP = pyridoxal phosphate...

In plants, algae, cyanobacteria and some other bacterial species 5-aminolevulinate (ALA) is synthesized from glutamate (1-6). This reaction sequence involves ligation of glutamate to a tRNA species and subsequent reduction to glutamate-l-semialdehyde (GSA) (7). The conversion of GSA to ALA is then catalysed by GSA-aminotransferase (E.C. S.4.3.8.) (2,8). Dependent on the requirement of pyridoxamine-phosphate (PAMP) or pyridoxal-phosphate (PALP) for activity, either 4,5-diaminovalerate (8-10) or 4,5-dioxovalerate (DOVA) (5,11,12) are proposed as intermediates. 

In rats fed a diet adequate in vitamin Bg, the fraction of total pyridoxal phosphate found in the nuclei of liver cells was 21%, and this increased to 39% in rats fed a vitamin Bg-deflcient diet, indicating a conservation of the vitamin in the nuclear compartment during deficiency. Pyridoxal phosphate in the cell nucleus is protein bound, and this protein has an apparent molecular mass of 50 to 55 kDa. Cells grown in the presence of 5 mM pyridoxine have a decreased glucocorticoid-dependent induction of enzymes such as tyrosine aminotransferase. Vitamin Bg regulates transcriptional activation of human glucocorticoid receptors in the HeLa cells. The modulatory role in transcription is not restricted to the glucocorticoid receptor but extends to other members of the steroid hormone super family. The intracellular concentration of PLP could have a profound influence on steroid hormone-induced... 

Use of aminotransferases Aminotransferases are pyridoxal 5 -phosphate-dependent enzymes that catalyze the reversible transfer of an amino group from an a-amino acid to an a-ketoacid in a two-step process. Of the many transaminases, aspartate aminotransferase (EC 2.6.1.1) is synthetically most useful since spontaneous decarboxylation of the generated oxaloacetate to pyruvate shifts the equilibrium to the product side. 

In vitro studies have been conducted to determine the effect of estrogens on kynurenine aminotransferase, which catalyzes the B(,-dependent transamination of kynurenine to ky-nurenic acid. Some estrogen conjugates (e.g.. c.stradiol disulfate and diethylstilbestrol sulfate) interfere with this transamination, apparently hy reversible inhibition of the aminotransfera.se apoenzyme. Apparently, the estrogen sulfate competes with pyridoxal S-phosphate for interaction with the apoenzyme. In conuast. free estradiol and estrone do not pos.sess this inhibitory property. 

The vitamin Bg group comprises three natural forms pyridoxine (pyridoxol) (PA/), pyridoxamine (PM), and pyridoxal (PL), which are 4-substituted 2-methyl-3-hydroxyl-5-hydroxymethyl pyridines (Figure 30-13). During metabolic conversions, each vitamer becomes phosphorylated at the 5-hydroxymethyl substituent. Although both pyridox-amine-5 -phosphate (PMP) and pyridoxal-S -phosphate (PLP, P-5 -P) interconvert as coenzyme forms during aminotransferase (transaminase)-catalyzed reactions, PLP is the coenzyme form that participates in the large number of Bg-dependent enzyme reactions. 

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