title: Dissecting the catalytic mechanism of protein-tyrosine phosphatases.
abstract: Protein-tyrosine phosphatases (PTPases) contain an evolutionarily conserved segment of 250 amino acids referred to as the PTPase catalytic domain. The recombinant PTPase domain from Yersinia enterocolitica enhances the rate of hydrolysis of p-nitrophenyl phosphate, a phosphate monoester, by approximately 10(11) over the non-enzyme-catalyzed rate by water. Specific amino acid residues responsible for the catalytic rate acceleration have been examined by site-directed mutagenesis. Our results suggest that Asp-356 (D356) and Glu-290 (E290) are the general acid and the general base catalysts responsible for Yersinia PTPase-catalyzed phosphate ester hydrolysis. The PTPase with both E290Q and D356N mutations shows no pH dependence for catalysis but displays a rate enhancement of 2.6 x 10(6), compared to the noncatalyzed hydrolysis of p-nitrophenyl phosphate by water. This rate enhancement probably occurs via transition-state stabilization. Our results suggest that all PTP-ases use a common mechanism that depends upon formation of a thiol-phosphate intermediate and general acid-general base catalysis.
authors.affiliation: Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109-0606.
authors.forename: Z Y
authors.initials: ZY
authors.lastname: Zhang
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: J E
authors.initials: JE
authors.lastname: Dixon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015008:Yersinia enterocolitica / Q000201:enzymology / Q000235:genetics
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases