The rate-determining step in pepsin-catalysed reactions, and evidence against an acyl-enzyme intermediate.
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.
D002247:Carbon Isotopes; D004151:Dipeptides; D004952:Esters; D005998:Glycine; D007700:Kinetics; D000432:Methanol; D010434:Pepsin A; D010455:Peptides; D010649:Phenylalanine; D014443:Tyrosine
D002247:Carbon Isotopes; D004151:Dipeptides; D004952:Esters; D010455:Peptides; D014443:Tyrosine; D010649:Phenylalanine; D010434:Pepsin A; D005998:Glycine; D000432:Methanol