Aspartic‐129 is an essential residue in the catalytic mechanism of the low M r phosphotyrosine protein phosphatase

FEBS Letters

Raugei, Giovanni¹  Stefani, Massimo¹  Taddei, Niccolò¹  Ramponi, Giampietro¹  Camici, Guido¹  Cirri, Paolo¹  Fiaschi, Tania¹  Chiarugi, Paola¹ 

[1] Department of Biochemical Sciences, University of Florence, Viale Morgagni 50, 50134 Florence, Italy

关键词: Low M r PTPase;    Phosphotyrosine protein phosphatase;    Low M r PTPase mutagenesis;    Low M r PTPase catalytic site;    PTPase;    phosphotyrosine protein phosphatase;    PNPP;    p-nitrophenylphosphate;    D129A;    aspartate-129 to alinine mutant;    IPTG;    isopropylthiogalattoside;    SDS-PAGE;    sodium dodecylsulfate polyacrylamide gel electrophoresis;    EDTA;    ethylenediaminotetraacetic acid;    UV;    ultraviolet;    NMR;    nuclear magnetic resonance;   

DOI  :  10.1016/0014-5793(94)00805-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The crystal structure of the bovine liver low M _r phosphotyrosine protein phosphatase suggests the involvement of aspartic acid-129 in enzyme catalysis. The Asp-129 to alanine mutant has been prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. The purified mutant elicited an highly reduced specific activity (about 0.04% of the activity of the wild-type) and a native-like fold, as judged by ¹H NMR spectroscopy. The kinetic analysis revealed that the mutant is able to bind the substrate and a competitive inhibitor, such as inorganic phosphate. Moreover, trapping experiments demonstrated it maintains the ability to form the E-P covalent complex. The Asp-129 to alanine mutant shows extremely reduced enzyme phosphorylation (k ₂) and dephosphorylation (k ₃) kinetic constant values as compared to the wild-type enzyme. The data reported indicate that aspartic acid-129 is likely to be involved both in the first step and in the rate-limiting step of the catalytic mechanism, i.e. the nucleophilic attack of the phosphorylated intermediate.

【 授权许可】

Unknown