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

Abstract

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.

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

“…The patterns of inhibition arising from the presence of acyl product and of chemical and stereochemical analogues of acyl product are reported. The results support and complement the preliminary studies of Greenwell et al (1969), and, together with work reported by Inouye & Fruton (1968) on the inhibition of pepsincatalysed reactions by the second type of product, support the pathway represented by eqn. (1) as operating for pepsin-catalysed hydrolysis, as well as for transpeptidation.…”

“…Both these studies were done at pH 2 and relate to inhibition by protonated N-acyl-amino acid. Greenwell et al (1969) have reported a value of 29mM for the inhibition of N-acetyl-L-phenylalanyl-L-phenylalanylglycine by the acyl product N-acetyl-L-phenylalanine at pH 2. The inhibition appeared to be non-competitive, 1971 244 though only a single inhibitor concentration was investigated.…”

“…N-Acetyl-L-phenylalanine. This was prepared as described by Greenwell et al (1969); it had m.p. 168-171°C and [a]20 +46.1°(c 2 in ethanol).…”

“…Classically, intermediates that follow the rate-determining step of a reaction can be detected by trapping experiments, where a reagent is added that reacts fast enough with the intermediate for one to observe product diversion with no change in overall reaction rate. Although the transpeptidation reaction itself is strictly an example of such product diversion, it was found by Greenwell, Knowles & Sharp (1969) that analogues of acyl-amino acids that would have allowed the transpeptidation reaction to be followed directly did not act as acceptors. Thus it was found that N-acetyl-L-phenylalanine ethyl ester cannot act as an 'amino group' acceptor in a transpeptidation process.…”

The pathway of pepsin-catalysed transpeptidation. Evidence for the reactive species being the anion of the acceptor molecule

“…N-Acetyl-L-[3H]phenylalanine. This compound was prepared from L-[3H]phenylalanine in the way described (for the unlabelled material) by Greenwell, Knowles & Sharp (1969); it had m.p. 169-171°C and [a]20 +46.10 (c 2 in ethanol).…”