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The inhibition of pepsin-catalysed reactions by structural and stereochemical product analogues.

ABSTRACT: 1. The inhibition of pepsin-catalysed hydrolysis of N-acetyl-l-phenylalanyl-l-phenylalanylglycine by products and product analogues was studied. 2. Inhibitors of the l-configuration give rise to linear non-competitive inhibition, whereas those of the d-configuration show linear competitive behaviour. 3. Non-competitive inhibition by the product N-acetyl-l-phenylalanine indicates an ordered release of products, which supports a common mechanism (involving an ;amino-enzyme') for pepsin-catalysed transpeptidation and hydrolysis reactions. 4. The differences in the types of inhibition caused by product analogues of the l- and d-series emphasize the stereospecificity of the binding of these inhibitors to free enzyme and to the putative amino-enzyme intermediate. 5. The results suggest that it is the anion of the acyl product that is released first in the hydrolytic reaction (see Kitson & Knowles, 1971).

SUBMITTER: Kitson TM

PROVIDER: S-EPMC1176768 | biostudies-other | 1971 Apr

REPOSITORIES: biostudies-other

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The inhibition of pepsin-catalysed reactions by products and product analogues. Kinetic evidence for ordered release of products.

Project description:1. The inhibition of pepsin-catalysed hydrolysis of N-acetyl-l-phenylalanyl-l-phenylalanylglycine by products and product analogues was studied. 2. The non-competitive nature of the inhibition by the product N-acetyl-l-phenylalanine confirms an ordered release of products, and points to a common mechanism (involving an amino-enzyme) for pepsin-catalysed transpeptidation and hydrolysis reactions. 3. N-Acetyl-l-phenylalanine ethyl ester is also a non-competitive inhibitor, but here the inhibition is of the ;dead-end' type. No ethanol is detectable in reaction mixtures, indicating that this ester cannot act as an amino group acceptor in a transpeptidation process. 4. The same is true for N-methanesulphonyl-l-phenylalanine methyl and methyl thiol esters. No methanethiol is liberated when the methyl thiol ester is present as an inhibitor of the hydrolytic reaction, and the hope that such a thiol ester would effectively trap the amino-enzyme was not fulfilled.

| S-EPMC1184643 | biostudies-other

The pH-dependence of pepsin-catalysed reactions.

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| S-EPMC1184642 | biostudies-other

SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis.

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The stereochemical course of reactions catalysed by the cellobiohydrolases produced by Talaromyces emersonii.

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Inhibition of human arginase I by substrate and product analogues.

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| S-EPMC2850953 | biostudies-literature

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

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A de novo approach to the synthesis of glycosylated methymycin analogues with structural and stereochemical diversity.

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The rate-determining step in pepsin-catalysed reactions, and evidence against an acyl-enzyme intermediate.

Project description:To delineate further the pathway of pepsin-catalysed reactions, three types of experiments were performed: (a) the enzyme-catalysed hydrolysis of a number of di- and tri-peptide substrates was studied with a view to observing the rate-determining breakdown of a common intermediate; (b) the interaction of pepsin with several possible substrates for which ;burst' kinetics might be expected was investigated; (c) attempts were made to trap a possible acyl-enzyme intermediate with [(14)C]methanol in both a hydrolytic reaction (with N-acetyl-l-phenylalanyl-l-phenylalanylglycine) and in a ;virtual' reaction (with N-acetyl-l-phenylalanine) under conditions where extensive hydrolysis or (18)O exchange is known to occur. It is concluded that (i) intermediates in pepsin-catalysed reactions (aside from the Michaelis complex) occur subsequently to the rate-determining transition state, and (ii) an acyl-enzyme intermediate, if such is formed, cannot be trapped with [(14)C]methanol in these systems.

| S-EPMC1184644 | biostudies-other

Use of progress curves to investigate product inhibition in enzyme-catalysed reactions. Application to the soluble mitochondrial adenosine triphosphatase.

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| S-EPMC2323848 | biostudies-literature

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