Malt carboxypeptidase catalyzed aminolysis reactions

Abstract

Malt carboxypeptidase catalyzes the formation of peptide bonds using N-benzoyl-arginine esters as acyl components and amino acid amides or amino acid methyl esters as nucleophiles. With seven different nucleophiles yields of 50–95% were obtained while no aminolysis was observed with H-Pro-NH2 and H-Glu-α-NH2. The enzyme is easily saturated with nucleophile indicating the formation of a complex between nucleophile and acyl-enzyme intermediate prior to deacylation. The nature of the side-chain of the amino acid esters used as nucleophiles strongly influences the apparent dissociation constant of this complex (KN(app)) while less dependence on the side-chain is observed with amino acid amides. KN(app) is drastically increased by the presence of phenylguanidine in the reaction medium suggesting a competition between this substance and the nucleophile for the same binding site. This site has previously been identified as the S1’ binding site of malt carboxypeptidase (Carlsberg Res. Commun. 48, 573–582 (1983)). The influence of the nucleophile H-Val-NH2 on the kinetic parameters for the cleavage of Nα-CBZ-Lys-p-nitrophenylester indicates that the acylation step is rate-limiting, and that H-Val-NH2 has two different modes of binding to the enzyme.