CARBOXYPEPTIDASE-Y-CATALYZED TRANSPEPTIDATION OF ESTERIFIED OLIGOPEPTIDES AND POLYPEPTIDES AND ITS USE FOR THE SPECIFIC CARBOXYL-TERMINAL LABELING OF PROTEINS

A systematic study of the specific carboxyl-terminal labeling of oligo- and polypeptides has been undertaken. It relies on chemical esterification of the peptidic substrate followed by carboxypeptidase Y-catalyzed transpeptidation at basic pH with an amino acid derivative as nucleophile. Two nucleophiles, glycine isobutyl ester (Gly-OiBu) and valine amide (Val-NH2), produced transpeptidation with a wide variety of short peptide substrates. Transpeptidation constants, i.e., concentrations of the nucleophile with which transpeptidation occurs at a velocity equal to that of the competing hydrolysis reaction, were in the range 1.2-21 mM for Val-NH2 and 20-240 mM for Gly-OiBu, depending on the esterified peptidic substrate used. Therefore, these compounds were selected as potential nucleophiles for a general method of protein labeling. Using tritiated Val-NH2 as nucleophile, labeling yields obtained with methylated porcine chymotrypsinogen A and methylated equine myoglobin as substrates were 31% and 39%, respectively. Several proteins, including Escherichia coli lysyl-tRNA synthetase, were labeled with tritiated Gly-OiBu or Val-NH2 and proteolyzed. In each case, a labeled peptide could be isolated and identified as the carboxyl-terminal peptide.