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  • Title: [Mutation of arginines near the active site Cys124 of human dual-specificity phosphatase and its effect on the enzymatic activity].
    Author: Wang YH, Zeng WY, Shi YY.
    Journal: Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai); 2003 Feb; 35(2):149-53. PubMed ID: 12545222.
    Abstract:
    To study the effect of three positively charged arginine residues near the active site Cys(124) of the human dual-specific phosphatase on the catalytic function, six VHR mutants R(125)L, R(130)L, R(130)K, R(130)L/S(131)A, R(158)K and R(158)L were obtained using QuikChange site-directed mutagenesis method. The recombinant plasmids containing mutant genes were transformed into the Escherichia coli strain BL21(DE3), and the expressed proteins were found to be water soluble after the induction of IPTG. The proteins with purity greater than 90% were obtained using Ni(2+) chelating affinity chromatography. The measurement of the steady-state kinetic parameters and arsenate inhibition constants K(i) of the enzyme and their mutants showed that the k(cat)/K(m) values of Arg(130) and Arg(158) mutants decreased, and K(i) values increased obviously compared with those of the wild enzyme. These results indicated that Arg(130) and Arg(158) were necessary for the enzymatic activity, and were probably related to the binding with the negatively charged phosphate group of the substrate. In addition, the slight difference for the k(cat) values between the R(130)L and R(130)L/S(131)A mutants suggested that Arg(130) mutation disrupted the hydrogen bond between Ser(131) and Cys(124). Furthermore, the arsenate binding affinity for R(125)L, R(130)L and R(158)L mutants was decreased, suggesting that positive charges in the side chains of these three arginine residues may be helpful for the binding of the enzyme to the substrate.
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