Synthetic and mechanistic studies of (2R,3S)-3-vinylmalic acid as a mechanism-based inhibitor of 3-isopropylmalate dehydrogenase

(2R,3S)-3-Vinylmalic acid (VM) was designed as a mechanism-based inhibitor of threo-3-isopropylmalate dehydrogenase (LPMDH), the rate-determining enzyme responsible for the penultimate step in the biosynthetic pathway of the essential amino acid L-leucine. The synthesis of VM was achieved in six steps from diethyl (R)-malate. Besides its weak activity as a substrate, VM was shown to be a mechanism-based inhibitor (K-I = 1.20 mM) for IPMDH as deduced from the time-dependent and kinetic analyses. 2-Oxo-3-pentenoate was identified as the enzyme reaction product of VM by GCMS analysis and H-1 NMR spectroscopy, but the supplemented 2-oxo-3-pentenoate was not, in contrast, inhibitive to the enzyme reaction. It seems likely from these results that an activated or nucleophilic amino acid of IPMDH by deprotonation during the ordinary enzyme reaction process participates in a strong interaction with the 2-oxo-3-pentenoate product, probably by the formation of a transient covalent bond, which in turn gives rise to inhibition of the enzyme reaction.