Nature of the rate-determining steps of the reaction catalyzed by the Yersinia protein-tyrosine phosphatase.

Abstract

Product inhibition and 18O exchange experiments suggest that the Yersinia protein-tyrosine phosphatase-catalyzed phosphate monoester hydrolysis proceeds through at least two different chemical steps, i.e. the formation and breakdown of a covalent phosphoenzyme intermediate. The pH dependence of kcat values is bell-shaped, with the apparent pKa derived from the acidic limb of the profile at 4.6 for both p-nitrophenyl phosphate and beta-naphthyl phosphate, whereas the apparent pKa derived from the basic limb of the profile is substrate-dependent, with apparent pKa values of 5.2 and 5.8 for p-nitrophenyl phosphate and beta-naphthyl phosphate, respectively. Twelve aryl phosphates with leaving groups having pKa values from approximately 7 to 10 are also examined as substrates at two pH values. At pH 4.0, the beta lg values is effectively zero, whereas at pH 7.5, a beta lg value of 0.16 is observed. Collectively, our results suggest that the rate-determining step under acidic conditions corresponds to the breakdown of the phosphoenzyme intermediate, whereas under more alkaline conditions, substrate effects also contribute to the rate-limiting step. A model is proposed for the mechanism of the Yersinia protein-tyrosine phosphatase-catalyzed reaction.