Evidence for Multiple Reactive Forms of Papain

The pH‐dependence of the second‐order rate constants of acylation and alkylation reactions of the –SH group of papain were determined by using neutral and charged reactants under identical conditions. From these pH rate profiles, in contrast to previous claims, different p K a values were obtained with different groups of reactants. In the case of charged reactants, like chloroacetate (p K a = 3.6) and arginine derivatives (p K a = 4.3), the p K a differences can be attributed to electrostatic effects. However, the fact that a p K a of 3.25 was found with methyl and ethyl bromoacetate, and a p K a of 4.0 was obtained with bromoacetamide and a number of neutral substrates, is inconsistent with the theories put forth hitherto for the meaning of such p K a values, because they all consider only one reactive enzyme form. The different p K a values obtained here with neutral reactants are explained in terms of various reactive papain forms. The perturbation of p K a by electrostatic effects was examined by reacting simple thiol compounds containing different charges, like 2‐mercaptoacetate, 2‐mercaptoethylamine, 2‐mercaptoethanol and glutathione, with the neutral chloroacetamide and with the negatively charged chloroacetate. Differences in p K a can be interpreted in terms of intramolecular and intermolecular electrostatic interactions.