James Z. Ginos

Inhibition of glutamate-aspartate transaminase by β-methylene-DL-aspartate☆

beta-Methylene-DL-aspartate, a new beta, gamma-unsaturated amino acid, is an irreversible inhibitor of soluble pig heart glutamate-aspartate transaminase (Ki approximately 3 mM with respect to the L-form; limiting rate constant for inactivation approximately 0.4 min-1). The new amino acid is the most specific inhibitor of glutamate-aspartate transaminase thus far studied. It does not inactivate pig heart glutamate-alanine transaminase, soluble rat kidney glutamine transaminase K, gamma-aminobutyrate transaminase (from Pseudomonas fluorescens), glutamate decarboxylase (Escherichia coli), snake venom L-amino acid oxidase, or hog kidney D-amino acid oxidase. In addition, the following enzymes were not inhibited by beta-methylene-DL-aspartate in rat tissue homogenates: gamma-aminobutyrate transaminase (brain), tyrosine transaminase (liver), glutamine transaminase L (liver), asparagine, transaminase (liver), ornithine transaminase (liver) or branch-chain transaminase(s) (kidney). Intraperitoneal injection of beta-methylene-DL-aspartate into mice decreased kidney and liver glutamate-aspartate transaminase activities but had no effect on liver glutamate-alanine transaminase activity.

Interaction of soluble pig heart glutamate-aspartate transaminase with various β,γ-unsaturated amino acids

β-Ethylidene-DL-aspartate (βEA) and β-methylene-DL-glutamate (βMG) were synthesized and tested as potential suicide inhibitors of soluble pig heart glutamate-aspartate transaminase (sGAT). βMG was found to be a) a substrate with a very low turnover number relative to glutamate and b) a competitive inhibitor with respect to aspartate (albeit with a large binding constant). At high concentrations βMG inactivated the enzyme but only very slowly. βEA was also found to be a substrate with a very low turnover number; it did not inactivate the enzyme (1 hr, 25°C) even at a high concentration. However, βEA was found to bind to the enzyme with an affinity comparable to that of aspartate and glutamate. β-Methylene-DL-aspartate (βMA) has been shown to rapidly inactivate glutamate-aspartate transaminase. Therefore, it appears that glutamate-aspartate transaminase can bind analogues of aspartate with alkene groups in the β position. The conjugated carbonyl groups of βMA and βEA will enhance Michael addition in comparison with that expected for vinylglycine. On the other hand, the presence of the methyl groups should reduce the electrophilicity of the double bond of βEA compared to βMA. This deactivation and/or steric hindrance to Michael attack may account for the inability of βEA to inactivate sGAT. Therefore, it may be possible to design selective suicide inhibitors of glutamate-aspartate transaminase with the following structure: HO2CC(CHX)CH(CO2H)NH2, where X is an electron-withdrawing group. Ideally, X would increase the reactivity of the double bond while affording a minimum of steric hindrance to susceptible enzyme-bound bases.