NATURE OF THE RATE-DETERMINING STEPS OF THE REACTION CATALYZED BY THEYERSINIA PROTEIN-TYROSINE-PHOSPHATASE

Product inhibition and O-18 exchange experiments suggest that the Yers inia protein-tyrosine phosphatase-catalyzed phosphate monoester hydrol ysis proceeds through at least two different chemical steps, i.e. the formation and breakdown of a covalent phosphoenzyme intermediate. The pH dependence of k(cat) values is bell-shaped, with the apparent pK(a) derived from the acidic limb of the profile at 4.6 for both p-nitroph enyl phosphate and beta-naphthyl phosphate, whereas the apparent pK(a) , derived from the basic limb of the profile is substrate dependent, w ith apparent pK(a) values of 5.2 and 5.8 for p-nitrophenyl phosphate a nd beta-naphthyl phosphate, respectively. Twelve aryl phosphates with leaving groups having pK(a) values from similar to 7 to 10 are also ex amined as substrates at two pH values. At pH 4.0, the beta(lg) value i s effectively zero, whereas at pH 7.5, a beta(lg) value of 0.16 is obs erved. Collectively, our results suggest that the rate-determining ste p under acidic conditions corresponds to the breakdown of the phosphoe nzyme 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-cataly zed reaction.