Acyl intermediates in penicillopepsin-catalysed reactions and a discussion of the mechanism of action of pepsins

Abstract

Penicillopepsin catalyses transpeptidation reactions involving the transfer of the N-terminal amino acids of suitable substrates via covalent acyl intermediates to acceptor peptides, usually the substrate. The major products obtained when Phe-Tyr-Thr-Pro-Lys-Ala and Met-Leu-Gly were used as substrates were Phe-Phe and Met-Met respectively. With Met-Leu-Gly the tetrapeptide Met-Met-Leu-Gly was observed as probable intermediate. Co-incubation of Leu-Tyr-Leu and Phe-Tyr-Thr-Pro-Lys-Ala led to the formation of Leu-Phe and Phe-Leu as well as Leu-Leu and Phe-Phe. No reaction was observed with tripeptides in which the first or second amino acid is glycine. It appears that two amino aicds with large hydrophobic residues are needed for the transpeptidation reaction. Nucleophilic compounds other than peptides, such as hydroxylamine, aliphatic alcohols and dinitrophenylhydrazine, were not acceptors for the acyl group. Leucine, phenylalanine and leucine methyl ester also had no effect on the reaction. The transpeptidation reaction proceeded readily at pH 3.6 and 4.7. At pH 6.0 the reaction was slow and at pH 1.9 little or no transpeptidation was observed. Porcine pepsin catalyses similar transpeptidation reactions. Sequence studies show that porcine pepsin and penicillopepsin are homologous. The present study also suggests that they have a very similar mechanism. Evidence available at this time indicates that the mechanism of these enzymes is complex and may be modulated by secondary substrate-enzyme interactions. A hypothesis is presented which proposes that pepsin-catalysed reactions proceed via different covalent intermediates (amino-intermediates or acylintermediates) depending on the nature of the substrate. The possibility that some reactions do not involve covalent intermediates is also discussed.