Mechanism of carboxypeptidase-Y-catalyzed reaction deduced from a pressure-dependence study.

The activation volumes for kcat of the carboxypeptidase-Y-catalyzed hydrolysis of ester substrates were slightly negative (-1 to -4 ml/mol), while those for peptide and depsi-peptide analog were highly positive (+10 to +27 ml/mol). These values and the contrasting pH dependences of these two groups of the substrates are explained by a mechanism involving three ionic states of the enzyme and the second stable intermediate (acyl-enzyme). Esters are mostly rate-controlled by the deacylation step and peptides are controlled by both the acylation and the deacylation steps. Pressure increase induced a partial shift of the rate-determining step. Reaction volumes for Km-1 of peptide and depsi-peptide analog showed large and positive values (+16 to +29 ml/mol) which reflects the electrostatic interaction in the substrate recognition by this enzyme.