Transaminases pyridoxal-5 -phosphate-dependent

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). 

The mechanism comprises two distinct halves, each able to occur without the other. This mechanism is followed for example by pyridoxal phosphate-dependent transaminases, the amino group being transferred from the donor substrate to the enzyme s prosthetic group and so, ultimately, to the acceptor substrate [40,41],... 

TABLE 29.1 Classification of Pyridoxal-5 -Phosphate-Dependent Transaminases Based on their Evolutionary Relationship ... 

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 2.6.1.42], 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... 

Amino acids NAD(P)H Pyridoxamine 5 -phosphate Pyruvate Amines Pyridoxal 5-phosphate dependent enzymes Dehydrogenases Transaminases Pyridoxal 5-phosphate dependent enzymes Amino acid decarboxylases... 

Indolmydn.—Previous evidence on the biosynthesis of indolmycin (88) in Strepto-myces griseus cultures accords with the pathway shown in Scheme 4. The first two steps in the pathway have been carried out using cell-free extracts of 5. griseus - and recent work has led to the isolation of two enzymes which can effect these transformations. The first, tryptophan transaminase, catalysed the pyridoxal phosphate-dependent transamination of L-tryptophan, but not D-trptophan, and in common with some other microbial transaminases, a-ketoglutarate was an efficient amino-group acceptor. L-Phenylalanine, tyrosine, and 3-methyltryptophan (this compound inhibited enzyme function) also underwent transamination. 

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. 

The experiments described earlier showed that in liver homogenates and extracts this reaction is brought about by transamination, which is an obligatory first step in the oxidation of tyrosine by such systems. The existence of su( h a transaminating system was already known (133, 134, 393), and the observed pyridoxal phosphate-dependence when transamination was was made rate-controlling (489) was in accordance with the known behavior of transaminases (c/. 482). 

The glycolytic pathway includes three such reactions glucose 6-phosphate isomer-ase (1,2-proton transfer), triose phosphate isomerase (1,2-proton transfer), and eno-lase (yS-elimination/dehydration). The tricarboxylic acid cycle includes four citrate synthase (Claisen condensation), aconitase (j5-elimination/dehydration followed by yS-addition/hydration), succinate dehydrogenase (hydride transfer initiated by a-proton abstraction), and fumarase (j5-elimination/dehydration). Many more reactions are found in diverse catabolic and anabolic pathways. Some enzyme-catalyzed proton abstraction reactions are facilitated by organic cofactors, e.g., pyridoxal phosphate-dependent enzymes such as amino acid racemases and transaminases and flavin cofactor-dependent enzymes such as acyl-C-A dehydrogenases others. 

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. 

These reactions involve the activities of transaminases and decarboxylases (see p. 210), and over 50 pyridoxal phosphate-dependent enzymes have been identified. In transamination, pyridoxal phosphate accepts the a-amino group of the amino acid to form pyridoxamine phosphate and a keto acid. The amino group of pyri-doxamine phosphate can be transferred to another keto acid, regenerating pyridoxal phosphate. The vitamin is believed to play a role in the absorption of amino acids from the intestine. 

Kynureninase (Figure 11.16) is a pyridoxal phosphate-dependent enzyme, and its activity falls markedly in vitamin deficiency, at least partly because it undergoes a slow mechanism-dependent inactivation that leaves catalytically inactive pyridoxamine phosphate at the active site of the enzyme. The enzyme can only be reactivated if there is an adequate supply of pyridoxal phosphate. This means that in vitamin deficiency there is a considerable accumulation of both hydroxykynurenine and kynurenine, sufficient to permit greater metabolic flux than usual through kynurenine transaminase, resulting in increased formation of kynurenic and xanthurenic acids. 

Various pyridoxal phosphate dependent enzymes compete with each other for the available pool of coenzyme. Thus the extent to which an enzyme is saturated with its coenzyme provides a means of assessing the adequacy of the body pool of coenzyme. This can be determined by measuring the activity of the enzyme before and after the activation of any apoenzyme present in the sample by incubation with pyridoxal phosphate added in vitro. Erythrocyte aspartate and alanine transaminases are both commonly used the results are usually expressed as an activation coefficient— the ratio of activity with added coenzyme to that without. 

The oxidative pathway of tryptophan metabolism is shown in Figure 3. Kynureninase is a pyridoxal phosphate-dependent enzyme, and in deficiency its activity is lower than that of tryptophan dioxygenase, so that there is an accumulation of hydroxy-kynurenine and kynurenine, resulting in greater metabolic flux through kynurenine transaminase and increased formation of kynurenic and xanthurenic acids. Kynureninase is exquisitely sensitive to vitamin Bg deficiency because it undergoes a slow inactivation as a result of catalysing the half-reaction of transamination instead of its normal reaction. The resultant enzyme with pyridoxamine phosphate at the catalytic site is catalytically inactive and can only be reactivated if there is an adequate concentration of pyridoxal phosphate to displace the pyridoxamine phosphate. 

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. 

A variety of studies have shown that 10% to 20% of the population of developed countries have marginal or inadequate stams, as assessed by erythrocyte transaminase activation coefficient (Section 9.5.36) or plasma pyridoxal phosphate (Section 9.5.1 Bender, 1989b). This may be sufficient to enhance the responsiveness of target tissues to steroid hormones (Section 9.3.3), and may be important in the induction and subsequent development of hormone-dependent cancer of the breast and prostate. Vitamin Be supplementation may be a useful adjunct to other therapy in these common cancers certainly, there is evidence that poor vitamin Be nutritional stams is associated with a poor prognosis in women with breast cancer. 

A number of studies have measured the activation of plasma transaminases by pyridoxal phosphate added in vitro however, it is difficult to interpret the results, because plasma transaminases arise largely accidentally, as a result of cell turnover, and the amount released will depend on tissue damage. Furthermore, there is a considerable amount of pyridoxal phosphate in plasma, largely associated with serum albumin, and the extent to which plasma transaminases are saturated will depend largely on the relative affinity of albumin and the enzyme concerned for the coenzyme, rather than reflecting the availability of pyridoxal phosphate for intracellular metabolism. Studies on erythrocyte transaminase activation coefficient are easier to interpret, because the extent to which the enzymes are saturated depends mainly on the availability of pyridoxal phosphate. 

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). 

Vitamin B Three substances are classed under the term pyridoxine or adermine pyridoxol, pyridoxal and pyridoxamine. Pyridoxine was isolated by various study groups in 1938. Its structure was described by Folkers and Kuhn in 1939. Pyridoxal and pyridoxamine were discovered by Snell in 1942. Pyridoxal phosphate and pyridoxamine phosphate are biologically active substances. Intestinal absorption of Bg is dose-dependent and not limited. In alcoholism, a deficiency of vitamin Bg is encountered in 20—30% of cases, whereas the respective percentage is 50—70% in alcoholic cirrhosis. Vitamin Bg is an important coenzyme for transaminases, which transfer amino groups from amino adds to keto acids. In this way, biochemical pathways between the dtiic acid cycle and carbohydrate and amino acid metabolisms are created. (104)... 

The late 1990s saw the development of an alternative methodology for the enzymatic resolution of racemic amines using transaminases. Transaminases are pyridoxal phosphate 50 dependent enzymes that catalyze the transfer of an amine group to a carbonyl compound (amine group acceptor), such as a ketone, aldehyde, or keto add (Figure 14.19). 

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. 

Pyridoxal phosphate is a co-enzyme for numerous enzymes, notably amino acid decarboxylases, amino acid transaminases, histaminase and probably diamine oxidase Ais.iw. As most of the evidence on which the mechanism of action of pyridoxal-dependent enzymes is based has been obtained from studies of the non-enzymic interaction of pyridoxal with amino acids, these non-enzymic reactions will be considered first in some detail. 

Although yeast cells were considered to incorporate up to 50% of wort amino acids directly into protein [62], analysis of the utilization of and labelled amino acids by brewers yeast show that negligible assimilation of complete amino acid occurs [63]. Thus, when amino acids enter the cell their amino groups are removed by a transaminase system and their carbon skeletons assimilated. Transaminases catalyse readily reversible reactions dependent upon the presence of the cofactor pyridoxal phosphate. The general mechanism of the reaction is depicted in Fig. 17.14. 

The first step in tyrosine oxidation is a transamination to form p-hydroxyphenylpyruvic acid. Several groups of investigators independently showed a dependence of tyrosine oxidation on the presence of a keto acid. Knox and LeMay-Knox showed that a-ketoglutarate is a specific partner in the transamination and that pyridoxal phosphate is a cofactor in this reaction. Partial resolution of the transaminase allowed a demonstration of parallel restoration of transaminase activity and over-all tyrosine oxidation by addition of pyridoxal phosphate. 

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