Effects of secondary binding by activator and inhibitor peptides on covalent intermediates of pig pepsin

Publisher Website
Google Scholar
Abstract
A number of peptides were found to increase the activity of pig pepsin towards small synthetic substrates. The activators increase transpeptidation of both the acyl-transfer and the amino-transfer types by as much as 45-fold. The effect on hydrolysis varies from inhibition to modest activation, but is always less than the effect on transpeptidation. The kinetics of substrate cleavage are the converse of non-competitive inhibition and show an increase in kcat. and no effect on Km values. Lineweaver-Burk plots of results obtained in the presence of the activators indicate a substrate activation at high substrate concentration. This appears to be a co-operative effect, since it is not observed in the absence of the activators. The activation is greatest at pH 4.7, less at pH3.4, and at pH2.0 is observable only with some of the activator peptides. The results show directly the effect of secondary binding on the catalytic efficiency of papsin. The most effective activators are those that are most hydrophobic. Binding in the secondary binding sites causes an increase in hydrophobicity in the catalytic site, which results in increased stability of the acyl and amino intermediates, and preferential reaction with acceptors other than water. The implication that the present results strength the case for a role of covalent intermediates in the hydrolysis of good substrates (high kcat. values) is discussed.