Inhibition of carboxypeptidase A by aldehyde and ketone substrate analogs

Abstract

DL-2-Benzyl-3-formylpropanoic acid is a competitive inhibitor of carboxypeptidase A with an apparent Ki of 0.48 .mu.M at pH 7.5 in 50 mM Tris buffer-0.5 M in sodium chloride with O-(trans-p-chlorocinnamoyl)-L-.beta.-phenyllactate as substrate. At pH 7.5 in deuterium oxide, DL-2-benzyl-3-formylpropanoic acid exists as an equilibrium mixture of 75% free aldehyde and 25% hydrated aldehyde. The species that binds to the enzyme may be either the free aldehyde or the hydrate. Therefore, the Ki of the species bound is significantly less than the observed Ki of 0.48 .mu.M. The alcohol and dioxolane analogs of this aldehyde, DL-2-benzyl-4-hydroxybutanoic acid and 2-benzyl-4,4-(ethylenedioxy)butanoic acid, are only weak inhibitors with Ki of 0.54 mM and 2 mM, respectively. The ketone, (.+-.)-3-(p-methoxybenzoyl)-2-benzylpropanoic acid ([.+-.)-I], had a Ki of 180 .mu.M, experimentally indistinguishable from that of the diastereomeric mixture of its alcohol analog 2-benzyl-4-hydroxy-4-(p-methoxyphenyl)butanoic acid, Ki = 190 .mu.M. The ketone (I) is not detectably hydrated (< 2%) at pH 7.5 in deuterium oxide. The hydratable aldehyde DL-2-benzyl-3-formylpropanoic acid may mimic an intermediate resembling the transition state for amide hydrolysis by carboxypeptidase A while the nonhydratable ketone does not do so.