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.

Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin.

“…Finally, conformational changes that involve the flap region ofthe molecule can begin to explain the acyl and amino transfers (24,28,29) that occur in transpeptidation reactions. Covalent intermediates are not likely in the aspartyl proteinase mechanism (7,14,15).…”

Acyl and amino intermediates in reactions catalysed by pig pepsin. Analysis of transpeptidation products

“…Analysis of reaction products by high-voltage electrophoresis. The reaction mixtures were separated as described previously (Takahashi & Hofmann, 1975) by high-voltage electrophoresis on Whatman 3MM paper in volatile buffer (pyridine/acetic acid/ water, 1:10:89, by vol.) at pH 3.6 (Bennett, 1967) and Methyl Green was added as a marker (Stevenson, 1971).…”

“…We have described (Takahashi et al, 1974;Takahashi & Hofmann, 1975) some initial studies of the action of pig pepsin and penicillopepsin on small substrates. We showed that the enzymes catalysed transpeptidation reactions that involved the transfer of an N-terminal amino acid via a covalent acyl intermediate.…”

“…On the basis of these experiments it was proposed that catalytic mechanisms of the action of pepsin described so far do not account for all the observed reactions. Takahashi & Hofmann (1975) proposed that the pathway of pepsinand penicillopepsin-catalysed reactions is determined by the nature of the substrate and that reactions may proceed via an amino intermediate, an acyl intermediate, or possibly no covalent intermediate. To evaluate further the significance of the covalent intermediate we decided to extend the study of substrates that show a transpeptidation of either aminoor acyl-transfer type.…”

The Mechanism of the Catalytic Action of Pepsin and Related Acid Proteinases

“…The possibility that acyl-enzyme intermediates are involved in pepsin catalysis has been raised anew, however, by the finding that porcine pepsin and penicillopepsin catalyze the transfer of the N-terminal leucyl residue of Leu-Tyr-Leu and Leu-Tyr-NH, to the substrate to form chromatographically detectable amounts of the Leu-Leu-Tyr-X products, which are then predominantly cleaved to yield Leu-Leu (30,89), as shown in the reactions below. The report that the action of pepsin on…”