P. Richman, J. Fruton
Nov 1, 1976
Citations
0
Influential Citations
6
Citations
Quality indicators
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abstract
Treatment of swine pepsin with acetylimidazole to acetylate approximately five of its 16 tyrosyl residues causes a significant enhancement of catalytic efficiency (kcat/Km) toward substrates such as dansyl-glycyl-glycyl-L-phenylalanyl-L-phenylalanine 3-(4-pyridyl)propyl ester and benzyloxy-carbonyl-(glycyl)n-p-nitroLphenylalnyl-Lphenylalanyl-L-tyrosine (where n = 0, 1,2). Stopped-flow kinetic studies, under conditions of enzyme excess, with the dansyl peptide have shown that, as with untreated pepsin, the rate-limiting step in the over-all catalytic process is associated with the decomposition of the first detectable enzyme-substrate complex, whose dissociation constant is approximately equal to the Km found in steady-state kinetic experiments. With substrates of the type benzoyl-(glycyl)n-nitro-L-phenylalanyl-L-tyrosine, an increase in the chain length of the peptide leads to an increase in the value of kcat/Km, supporting the view that secondary enzyme-substrate interactions may produce at the extended active site conformational changes that are reflected in higher catalytic efficiency. This effect is more marked with acetyl-pepsin than with untreated pepsin, and suggests that the conformational mobility of the active site is increased by partial acetylation. Acetyl-pepsin is less effective than untreated pepsin in catalyzing transpeptidation reactions in which acetyl-L-phenylalanyl-L-tyrosine and benzyloxycarbonyl-(glycyl)n-p-nitro-L-phenylalanine are the reactants; this finding is consistent with the more rapid hydrolysis of the product of transpeptidation.