| Abstract: |
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. © 1985. |
