[Mutation of arginines near the active site Cys124 of human dual-specificity phosphatase and its effect on the enzymatic activity].
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.
Wang
Ying-Hua
YH
Key Laboratory of Structural Biology, School of Life Sciences, University of Science and Technology of China, Hefei 230027, China.
Zeng
Wang-Yong
WY
Shi
Yun-Yu
YY
D001120:Arginine / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000235:genetics*; D054640:Dual Specificity Phosphatase 3; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
D011994:Recombinant Proteins; D001120:Arginine; C517025:DUSP3 protein, human; D054640:Dual Specificity Phosphatase 3; D017027:Protein Tyrosine Phosphatases; D003545:Cysteine