IDENTIFICATION OF THE CATALYTIC HISTIDINE RESIDUE PARTICIPATING IN THE CHARGE-RELAY SYSTEM OF CARBOXYPEPTIDASE-Y

The essential histidine residue of carboxypeptidase Y (CPY) was modifi ed by a site-specific reagent, a chloromethyl-ketone derivative of ben zyloxycarbonyl-L-phenylalanine. The single modified histidine residue was converted to N-T-carboxymethyl histidine (cmHis) upon performic ac id oxidation. A peptide containing cmHis was isolated from the tryptic -thermolytic digest. Based on the amino acid composition and sequence analysis, the peptide is shown to be Val-Phe-Asp-Gly-Gly-cmHis-MetO(2) -Val-Pro, which was derived from CPY cleaved by trypsin at Arg 391 and thermolysin at Phe 401, and thus His 397 was modified. This histidine residue has been implicated previously by X-ray analysis to participa te in the charge-relay system of CPY.