Inhibition of carboxypeptidase A by aldehyde and ketone substrate analogues

DL-2-Benzyl-3- formylpropanoic acid ( XIVb ) is a competitive inhibitor of carboxypeptidase A with an apparent Ki of 0.48 microM 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 microM. The alcohol and dioxolane analogues of this aldehyde, DL-2-benzyl-4-hydroxybutanoic acid (XI) and 2-benzyl-4,4-(ethylenedioxy)butanoic acid ( XXVII ), are only weak inhibitors with Ki's of 0.54 mM and 2 mM, respectively. The ketone, (+/-)-3-(p- methoxybenzoyl )-2- benzylpropanoic acid [(+/-)-I; Sugimoto , T., & Kaiser, E. T. (1978) J. Am. Chem. Soc. 100, 7750-7751], was found to have a Ki of 180 microM, experimentally indistinguishable from that of the diastereomeric mixture of its alcohol analogue 2-benzyl-4-hydroxy-4-(p-methoxyphenyl)butanoic acid (III), Ki = 190 microM. The ketone (I) is not detectably hydrated (less than 2%) at pH 7.5 in deuterium oxide. These results suggest that 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.