Mechanisms of carboxymethylation of bovine pancreatic nucleases by haloacetates and tosylglycolate.

Certain histidine residues in bovine pancreatic ribonuclease and deoxyribonuclease react with iodoacetate about 1UOO times faster than free histidine reacts. The mechanisms of these facilitated, active-site-directed reactions were investigated by study of the kinetics of carboxymethylation with reagents with varied leaving groups: chloride, bromide, iodide, and tosylate. With ribonuclease, the rate of reaction gave hyperbolic dependence on the concentration of reagent. The data fit a Michaelis-Menten mechanism, with dissociation constants of 8 to 24 rnM for the various reagents. The similar magnitudes of the constants lead to the conclusion that the reagents bind (reversibly) in a common way to the enzyme. The pseudo bimolecular rate constants for reaction (after correction for the differing inherent reactivities of the reagents) were also similar, indicating that the leaving group is not very important in the mechanism. These studies support the proposal that the reaction of haloacetates with ribonuclease is facilitated by ionicattraction of the carboxylate ion by a protonated imidazole while an unprotonated imidazole displaces the halide ion. In contrast, the kinetics of carboxymethylation of the Cu2+Tris complex of deoxyribonuclease showed that the reagents bound loosely, with dissociation constants of 130 xxl~ or larger. Furthermore, the corrected, pseudo bimolecular rate constants increased as the size of the leaving group decreased. The larger groups may be more sterically hindered in the reaction; alternatively, the smaller groups may be attracted more to the electrophilic Cu2+. In either case, during carboxymethylation, both the carboxylate ion and the leaving group interact with DNase. While the carboxymethylation of RNase may be facilitated by the precise orientation of the reagent, the carboxymethylation of DNase may be facilitated by electrophilic catalysis. Tosylglycolate (carboxymethyl p-toluenesulfonate) was prepared and tested as an analog of the haloacetates. The reagent was about one-eighth as reactive as iodoacetate with a pyridine base and reacted with the -SH group of