Mechanistic studies on carboxypeptidase A from goat pancreas: Role of arginine residue at the active site

Abstract

Chemical modification of carboxypeptidase Ag 1 from goat pancreas with phenylglyoxal or ninhydrin led to a loss of enzymatic activity. The inactivation by phenylglyoxal in 200 m m N-ethylmorpholine, 200 m m sodium chloride buffer, pH 8.0, or in 300 m m borate buffer, pH 8.0, followed pseudo-first-order kinetics at all concentrations of the modifier. The reaction order with respect to phenylglyoxal was 1.68 and 0.81 in 200 m m N-ethylmorpholine, 200 m m NaCl buffer and 300 m m borate buffer, pH 8.0, respectively, indicating modification of single arginine residue per mole of enzyme. The kinetic data were supported by amino acid analysis of modified enzyme, which also showed the modification of single arginine residue per mole of the enzyme. The modified enzyme had an absorption maximum at 250 nm, and quantification of the increase in absorbance showed modification of single arginine residue. Modification of arginine residue was protected by β-phenylpropionic acid, thus suggesting involvement of an arginine residue at or near the active site of the enzyme.