| Abstract: | The possible role of histidine residues in the catalytic function of carboxypeptidase Y from bakers' yeast has been investigated using site-specific reagents. Among the reagents tested, benzyloxy-L-phenylalanylchloromethane (Z-PheCH2Cl) was the most powerful inhibitor of the enzyme. It irreversibly inactivated both the peptidase and esterase activities with an apparent second order rate constant of 3.8 M-minus 1 S-minus 1; the D isomer caused essentially no effect on either activity. Inhibition by L-Z-PheCH2Cl, the reaction retarded by certain competitive inhibitors of the enzyme. Using radioactive L-Z-PheCH2Cl, the reaction with the enzyme was shown to be essentially stoichiometric. Diisopropylphosphorofluoridate (iPr2PF)-inactivated enzyme failed to react with Z-PheCH2Cl, and conversely, the Z-PheCH2Cl-inhibited enzyme failed to react with radioactive iPr2PF. Amino acid analyses of the Z-PheCH2Cl-inactivated enzyme revealed the loss of essentially 1 residue, with a concomitant yield of a 0.62 residue of N-t-carboxymethylhistidine. Since carboxypeptidase Y has a reactive serine at its active center, we concluded from these results that the mechanism involves a charge-relay system in the hydrolysis of peptide and ester substrates, as in chymotrypsin. An -SH group of carboxypeptidase Y was not affected during the reaction with L-Z-PheCH2Cl. The generic name "serine carboxypeptidase" has been proposed for carboxypeptidase Y and for the iPr2PF-sensitive carboxypeptidases from plants, molds, and animal tissues, in order to distinguish them from "metal carboxypeptidase" to which carboxypeptidase A (EC 3.4.12.2) and B (EC 3.4.12.3) belong. |
