title: 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.
authors.affiliation: Department of Biological Chemistry, Medical School, Walther Cancer Institute, Ann Arbor, Michigan.
authors.forename: Z Y
authors.initials: ZY
authors.lastname: Zhang
authors.forename: W P
authors.initials: WP
authors.lastname: Malachowski
authors.forename: R L
authors.initials: RL
authors.lastname: Van Etten
authors.forename: J E
authors.initials: JE
authors.lastname: Dixon
mesh_terms: D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D013379:Substrate Specificity; D015007:Yersinia / Q000201:enzymology*
chemical_list: D010103:Oxygen Isotopes; D010710:Phosphates; D010748:Phosphopeptides; D017027:Protein Tyrosine Phosphatases