( Key Laboratory of Structural Biology, School of Life Sciences, University of Science and Technology of China, Hefei 230027, China )

Abstract To study the effect of three positively charged arginine residues near the active site Cys¹²⁴ of the human dual-specific phosphatase on the catalytic function, six VHR mutants R¹²⁵L, R¹³⁰L, R¹³⁰K, R¹³⁰L/S¹³¹A, R¹⁵⁸K and R¹⁵⁸L 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 Ni²⁺ chelating affinity chromatography. The measurement of the steady-state kinetic parameters and arsenate inhibition constants Ki of the enzyme and their mutants showed that the kcat/Km values of Arg¹³⁰ and Arg¹⁵⁸ mutants decreased, and Ki values increased obviously compared with those of the wild enzyme. These results indicated that Arg¹³⁰ and Arg¹⁵⁸ 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 kcat values between the R¹³⁰L and R¹³⁰L/S¹³¹A mutants suggested that Arg¹³⁰ mutation disrupted the hydrogen bond between Ser¹³¹ and Cys¹²⁴. Furthermore, the arsenate binding affinity for R¹²⁵L, R¹³⁰L and R¹⁵⁸L 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.