Effects of substituents on the rates of deacylation of substituted benzoyl papains. Role of a carboxylate residue in the catalytic mechanism

Abstract

The effect of ring substitutents on the rates of deacylation of 8 meta- and para-substituted benzoyl papains [EC 3.4.22.2] was evaluated. The rate constants were found to depend upon a single ionizing group of pKa = 4.2-4.3, and to decrease by a factor of approximately 2.2 when measured in 94% D2O/H2O. The rates of deacylation are increased greatly by electron-withdrawing groups on the benzene ring. The Hammett .rho. value is 2.74 .+-. 0.32. A plot of the rate constants for deacylation of the benzoyl papains against the corresponding constants for substituted benzoyl chymotrypsins generates a straight line of slope 1.0. This result suggests a very similar distribution of charge on the benzoyl moiety in the transition state for the 2 enzymes, which is interpreted in terms of the net charge of the transition state for the deacylation of non-specific acyl papains being equal to -1, with the general base catalyzed assistance to the attack of water on the acyl enzyme being provided by the negatively charged Asp-158 rather than by the neutral Asn-175-His-159 hydrogen bond network. This result together with a survey of literature data suggests that the role of Asp-158 in papain catalysis was underestimated. The evidence advanced to date in support of the proposition that an imidazolium-150- to be insufficient to decide the issue.