Proton-Relay System of Carboxypeptidase Y as a Sole Catalytic Site: Studies on Mutagenic Replacement of His 397

Abstract

His397 was replaced with alanine by site-directed mutagenesis of the cloned PRC1 gene in order to confirm the role of this residue in the proton-relay system of carboxypeptidase Y (CPY). The expressed and purified H397A showed a CD spectrum almost identical to that of the wild type enzyme, but its heat stability and conformation on heating differed somewhat. Kinetic analysis showed that the k_cat values of the purified H397A toward the peptide substrates, Z-Phe-Leu and Z-Gly-Phe, were reduced to approximately 4×10^-5-fold, whereas the K_m values remained almost unchanged. The activity of the H397A preparation with the ester substrate, Ac-Phe-OEt, was negligible. The low activity of our H397A was lost on treatment with DFP and Z-Phe-CH₂Cl, site-specific inhibitors, respectively, for Ser146 and His397, and with the HgCl₂ and PCMB, SH-reagents for Cys341. After treatment with these inhibitors, the k_cat value for the H397A preparation toward Z-Phe-Leu decreased 1×10³-fold or more. The value was approximately 10^-8 for the wild type enzyme. This level of activity is 10³-fold lower than the reported value for the same mutant of CPY [Carlsberg Res. Commun. 54, 165-171 (1989)], and more than 10-fold lower than the values for the corresponding His-to-Ala mutants of trypsin [J. Am. Chem. Soc. 114, 1784-1790 (1992)] and subtilisin [Nature 332, 564-568 (1988)]. These findings, together with the pH profiles and chromatographic behavior, are evidence that the low activity of the H397A preparation is due to contamination by wild type CPY. The decreased k_cat value of our H397A mutant is the lowest reported among the corresponding histidine mutants of serine proteases. We conclude that the proton-relay system composed of Ser146 and His397 is the sole catalytic center of CPY, and that its destruction leads to complete inactivation.