Enzymatic properties of an acid carboxypeptidase from Aspergillus niger var. macrosporus.

Acid carboxypeptidase (peptidyl-L-amino-acid hydrolase, EC 3.4.16.1) isolated from Aspergillus niger var. macrosporus was investigated in regard to its kinetic parameters for two synthetic substrates. The optimum pH of peptidase activity toward Z-Glu-Tyr-OH was pH 3.0 Km and kappa cat values were 4.0 . 10(-3) M and 270 s-1 at pH 3.0 and 30 degrees C. The optimal pH of esterase activity toward Bz-Arg-OEt was pH 5.2 Km and kappa cat for esterolytic activity were 6.1 . 10(-4) M and 1500 s-1 at pH 5.0 and 30 degrees C. The enzyme released expected amino acids sequentially from the carboxyl ends of S-beta-aminoethylated ribonuclease A and the B-chain of oxidized insulin, demonstrating carboxypeptidase activity of the enzyme. The enzyme was inactivated by diisopropylphosphorofluoridate and phenyl-methanesulfonyl fluoride. In the reaction with [14C]diisopropylphosphorofluoridate, the amount of [14C]diisopropylphosphoryl group incorporated into the enzyme in complete inactivation was estimated as 2 mol/mol enzyme.