Aspartate transaminase mechanisms

Minor increases in serum transaminases without evidence of liver dysfunction are common in patients receiving standard heparin or low molecular weight heparin given therapeutically or prophylactically (60,61). This rise is more pronounced for alanine transaminase than for aspartate transaminase and occurs after 5-10 days of heparin treatment (61). The source of heparin has no relation to the development of raised transaminases. After withdrawal of heparin and sometimes even in spite of continued treatment (60,61), the transaminases return to normal (62,63). The mechanism of these increases has not been elucidated. A concomitant increase in gamma-glutamyl transpeptidase activity has been described in some patients (64). 

Confirmation of the molecular structure of the enzyme-inactivator adduct has been obtained for few modified PLP-dependent enzymes. In the case of the reaction of aspartate transaminase (aspartate aminotransferase) with L-serine 0-sulfate, the surprising result thus obtained by Metzler and co-workers has forced reevaluation of the mechanism of similar inactivators (Ueno et al., 1982). Conventional wisdom argued that the reaction should involve elimination of sulfate from the inactivator followed by addition of an enzyme nucleophile to the resulting double bond (Fig. 8). When subjected to high pH, however, the inactivated enzyme releases a yellow PLP adduct which has been identified as the aldol product of the cofactor and C-3 of pyruvate (9, Fig. 9) as previously prepared by... 

Fig. 8. Original mechanism proposed for inactivation of aspartate transaminase by serine 0-sulfate.

Obviously, the elucidation of the enzymic mechanism required the preliminary purification of at least one of the transaminases. An 85-90% pure glutamic aspartic transaminase was obtained and found to contain 2 moles of pyridoxal phosphate per mole of enzyme. But pyridoxal is not the active coenzyme. Gunsalus, Bellamy, and Umbreit discovered that the addition of pyridoxal to a culture medium of a strain of Streptococcus faecalis grown on a pyri-doxal-deficient medium has little effect on the ability of the bacteria to decarboxylate tyrosine. When the culture was supplemented with pyridoxal and adenosine triphosphate, or with phosphorylated derivatives of pyridoxal, the tyrosine decarboxylation activity was greatly enhanced. It was later established that... 

If this were indeed the mechanism of action of pyridoxal phosphate, then one would think that either pyridoxal or pyridoxamine phosphate would be active in catalyzing the enzymatic transamination. In one case recently with a highly purified glutamic aspartic transaminase only pyridoxal phosphate was capable of reconstituting the system. 

Mechanisms of action of pyridoxal phosphate (a) in glutamate-oxaloacetate transaminase, and (b) in aspartate /3-decarboxylase. 

L-Amino acid transaminases are ubiquitous in nature and are involved, be it directly or indirectly, in the biosynthesis of most natural amino acids. All three common types of the enzyme, aspartate, aromatic, and branched chain transaminases require pyridoxal 5 -phosphate as cofactor, covalently bound to the enzyme through the formation of a Schiff base with the e-amino group of a lysine side chain. The reaction mechanism is well understood, with the enzyme shuttling between pyridoxal and pyridoxamine forms [39]. With broad substrate specificity and no requirement for external cofactor regeneration, transaminases have appropriate characteristics to function as commercial biocatalysts. The overall transformation is comprised of the transfer of an amino group from a donor, usually aspartic or glutamic acids, to an a-keto acid (Scheme 15). In most cases, the equilibrium constant is approximately 1. 

D-Amino acid transaminases have been less well characterized but proceed by a similar catalytic mechanism and show similar potential as effective biocatalysts. Further strain development has incorporated amino acid racemases, enabling complete utilization of racemic amino donors. Thus, L-aspartic acid can be used as the n-amino acid donor through use of aspartate racemase within the system. 

Importantly, its precise mechanism of its anticonvulsant action is still not fully understood. However, it has been duly advocated that its administration specifically inhibits GABA-transaminase, and thereby enhancing the concentration of cerebral GABA. It has also been observed that a few other straight-chain saturated fatty acids i.e., lower fatty acids, such as propanoic acid, butyric acid, and pentanoic acid which are devoid of anticonvulsant characteristic features are relatively more potent and efficacious inhibitors of GABA-transaminase than is valproic acid. Furthermore, it has been adequately substantiated that there exists a rather stronger correlation between the anticonvulsant potency of valproate and other branched-chain fatty acids besides, their capability to minimise the prevailing concentration of cerebral aspartic acid (an amino acid). 

Centrilobular hepatic necrosis by single doses of coumarin (1,2-benzopyrone, ds-o-coumarinic acid lactone) have been reported in the rat (Lake 1984, Lake et al. 1989, Fentem et al. 1992), whereas chronic administration resulted in bile duct lesions (Hagan etal. 1967, Cohen 1979, Evans etal. 1989). The mechanism of acute coumarin-induced hepatotoxicity in the rat has been investigated by comparing the effects of coumarin with those of a number of methyl-substituted coumarin derivatives (Lake etal. 1994). Coumarin administration produced dose-related hepatic necrosis and a marked elevation of plasma alanine aminotransferase and aspartate aminotransferase activities. In contrast, non of the coumarin derivatives examined produced either hepatic necrosis or elevated plasma transaminase activities. Coumarin reduced hepatic microsomal ethylmorphine N-demethylase and 7-ethoxycoumarin 0-deethylase activities, whereas one or both mixed function oxidases appeared to be induced by treatment with 3,4-dimethylcoumarin, 4-methylcoumarin, 3-methyloctahydrocoumarin and 4-methyloctahydrocoumarin. These results provides an evidence that acute coumarin-induced hepatotoxicity in the rat is due to the formation of a coumarin 3,4-epoxide intermediate. 

Chloroalanine has been found to be an irreversible inhibitor of the pyridoxal phosphate-linked yS-aspartate decarboxylase/ aspartate aminotransferase/ and alanine racemase. The mechanism of inhibition is shown above by Eq. (7) (the sulfate reacts in the same manner) amino-ethane sulfonate irreversibly inhibits pyridoxal phosphate-linked GABA transaminase and L-serine-O-sulfate irreversibly inhibits aspartate aminotransferase. ... 

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