The pH-dependence of pepsin-catalysed reactions

Abstract

1. The pH-dependence of the pepsin-catalysed hydrolysis of three peptide substrates was studied by using a method for the continuous monitoring of the formation of ninhydrin-positive products. 2. Two peptide acid substrates, N-acetyl-l-phenylalanyl-l-phenylalanine and N-acetyl-l-phenylalanyl-l-phenylalanyl-glycine, show apparent pKa values of 1·1 and 3·5 in the plots of k0/Km versus pH. By contrast a neutral substrate, N-acetyl-l-phenylalanyl-l-phenylalanine amide, shows apparent pKa values of 1·0 and 4·7. 3. Together with the data of the preceding paper (Knowles, Sharp & Greenwell, 1969), these results are taken to indicate that the rate of pepsin-catalysed hydrolysis is controlled by the ionization of two groups, which on the free enzyme have apparent pKa values of 1·0 and 4·7. It is apparent that the anions of peptide acid substrates are not perceptibly bound to the enzyme, resulting in apparent pKa values of 3·5 for the dependence of k0/Km for these materials.