title: Asp129 of low molecular weight protein tyrosine phosphatase is involved in leaving group protonation.
abstract: Site-directed mutagenesis was used to explore the functions of a number of acidic residues of bovine low molecular weight protein tyrosine phosphatase. Residues Asp-129, Asp-56, and Asp-92 were mutated to Ala or Asn. The mutant enzymes D56A, D56N, and D92A showed no significant changes in Vmax values, although they did exhibit significantly altered Km values. In contrast, the D129A mutant enzyme exhibited a greater than 2000-fold reduction in Vmax, using p-nitrophenyl phosphate as a substrate. The Vmax values of D129A also exhibited a leaving group dependence, an altered solvent isotope effect of VmaxH/VmaxD of 0.78, and a lack of dependence on the presence of alternative phosphate acceptor alcohols, all properties that distinguish this mutant from wild type enzyme. The differences are due to a change of the rate-limiting step of the catalytic reaction. Asp-129 is concluded to be the proton donor to the leaving group in the phosphorylation step, and its mutation to alanine results in a reduced Vmax value and a change in the rate-limiting step of the catalysis from dephosphorylation to phosphorylation. Mechanistic considerations suggest that other phosphotyrosyl phosphatases having cysteine at the active site may be expected to have a similar requirement for a proton donor.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Z
authors.initials: Z
authors.lastname: Zhang
authors.forename: E
authors.initials: E
authors.lastname: Harms
authors.forename: R L
authors.initials: RL
authors.lastname: Van Etten
mesh_terms: D000818:Animals; D001224:Aspartic Acid; D001483:Base Sequence; D002417:Cattle; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry*; D011522:Protons
chemical_list: D011522:Protons; D001224:Aspartic Acid; D017027:Protein Tyrosine Phosphatases