Proton Inventory of Carboxypeptidase Y-Catalyzed Reactions

The partial solvent isotope effect of D2O on the carboxypeptidase Y-catalyzed hydrolysis of a peptide (3-(2-furyl)acryloyl-l-phenylalanyl-l-phenylalanine) and an ester (3-(2-furyl)acryloyl-l-phenylalanine ethyl ester) was investigated. The peptide hydrolysis showed a smaller kinetic isotope effect regarding the catalytic rate constant (kcat) and the second-order rate constant (kcat⁄Km). kcat Varied linearly on the heavy-water content, indicating that one proton is essentially transferred during the rate-determining step. On the other hand, an ester hydrolysis showed a larger, nonlinear effect, indicating the possibility of a transfer of two or more protons in the rate-determining step of the ester hydrolysis, possibly the deacylation step.