Zhang Z, Harms E, Van Etten R L
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
J Biol Chem. 1994 Oct 21;269(42):25947-50.
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.
采用定点诱变技术来探究牛低分子量蛋白酪氨酸磷酸酶中多个酸性残基的功能。将天冬氨酸残基-129、天冬氨酸残基-56和天冬氨酸残基-92突变为丙氨酸或天冬酰胺。突变酶D56A、D56N和D92A的最大反应速度（Vmax）值无显著变化，不过它们的米氏常数（Km）值确实有显著改变。相比之下，以对硝基苯磷酸酯作为底物时，D129A突变酶的Vmax降低了2000多倍。D129A的Vmax值还表现出离去基团依赖性、VmaxH/VmaxD的溶剂同位素效应改变为0.78以及对替代磷酸受体醇的存在不依赖，所有这些特性都将该突变体与野生型酶区分开来。这些差异是由于催化反应限速步骤的改变。得出结论，天冬氨酸残基-129是磷酸化步骤中离去基团的质子供体，将其突变为丙氨酸会导致Vmax值降低，并且催化的限速步骤从去磷酸化转变为磷酸化。机理方面的考虑表明，活性位点含有半胱氨酸的其他磷酸酪氨酸磷酸酶可能对质子供体有类似的需求。
