MALT CARBOXYPEPTIDASE CATALYZED AMINOLYSlS REACTIONS

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 5095% were obtained while no aminolysis was observed with H-Pro-NH2 and H-GIu-a-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 ofphenylguanidine 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 S~ binding site of malt carboxypeptidase (Carlsberg Res. Commun. 48,573-582 (1983)). The influence of the nucleophile H-VaI-NH2 on the kinetic parameters for the cleavage of N,-CBZ-Lys-p-nitrophenylesler indicates that the acylation step is rate-limiting, and that H-Val-NH2 has two different modes of binding to the enzyme.