Inhibition of aspartate aminotransferase by d-hydrazinosucinate: comparison with l-hydrazinosuccinate

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l-Hydrazinosuccinate has been reported to be a slow- and tight-binding inhibitor of aspartate aminotransferase ( l-aspartate:2-oxoglutarate aminotransferase, EC 2.6.11) and to interact with the enzyme via a reaction of two consecutive steps. The present work examined the effects of d-hydrazinosuccinate on the same enzyme for comparison. d-Hydrazinosuccinate showed a potent inhibition in a slow-binding manner: transmination became slower with time when the reaction was initiated by the addition of enzyme to a mixture of the assay components and d-hydrazinosuccinate, while the reaction was initially very slow and became faster with time when the enzyme was preincubated with the inhibitor before the initiation of reaction. Analysis of the time-course of interaction of the enzyme with d-hydrazinosuccinate suggested a reversible single-step reaction mechanism and gave an inhibition constant of approx. 3 nM, in contrast to the two-step mechanism, and a much lower inhibition constant of 0.2 nM for l-hydrazinosuccinate. Comparison of the rate constants for the reaction steps in the interaction of the enzyme with d- and l-enantiomers confirmed that the difference in the reaction mechanism was mainly responsible for the stronger inhibition by the l-enantiomer. Spectral studies showed that d- and l-hydrazinosuccinate both produced complexes with the enzyme probably in the form of aldimine, and thereafter only the complex with l-hydrazinosuccinate further changed to another species more slowly, consistent with the two-step mechanism. The configuration of the hydrazino group is therefore crucial for the conversion of aldimine complexes to more tightly bound complexes.