Effect of Enzyme–Substrate Interactions Away from the Reaction Site on Carboxypeptidase A Catalysis

The kinetics of 14 peptide substrates of carboxypeptidase A have been studied for the purpose of evaluating P1–P3/S1–S3interactions. It was found that the amide group at P1–P2is required for efficient catalysis. This observation is consistent with previously proposed hydrogen bonding interactions, based on crystallographic data, between the P1NH and Tyr-248 and between the P2carbonyl oxygen and Arg-71. In contrast, substitution of the benzamido amide group (at P2–P3) ofN-benzoylglycylglycyl-L-phenylalanine by –CH2CH2– resulted in more effective catalysis. In this case hydrophobic interactions are important in the ground state and in the transition state of the rate-determining step.