人双专一性磷酸酶活性位点Cys^124附近精氨酸突变及功能

为研究人双专一性磷酸酶活性位点Cys12 4 附近 3个带正电的精氨酸对酶催化功能的影响 ,用QuikChange定点突变方法获得 6个突变体 :R12 5L、R130 L、R130 K、R130 L/S131A、R158K和R158L。将含突变基因的重组质粒转化大肠杆菌菌株BL2 1(DE3) ,经IPTG诱导表达获得的目的蛋白质均以可溶形式存在。通过镍离子亲和层析纯化得到纯度大于 90 %的蛋白质。对人痘苗病毒H1相关磷酸酶 (VHR)及其突变体进行稳态动力学参数和竞争性抑制常数Ki 的测定 ,结果显示上述Arg130 和Arg158突变体的kcat/Km 值都较野生型有大幅度下降 ,而Ki 值有明显上升 ,表明 130和 15 8位的精氨酸是VHR活性所必需 ,而且可能与底物上带负电的磷酸基团结合有关。另外 ,单突变体R130 L和双突变体R130 L/S131A之间的kcat值相差很小 ,提示Arg130 单点突变后可能破坏了Ser131与Cys12 4 间的氢键。再者 ,R12 5L、R130 L和R158L突变体都降低了砷酸盐结合亲和性 ,暗示这 3个精氨酸残基侧链上的正电荷可能有助于底物与酶的结合。

Mutation of Arginines Near the Active Site Cys 124 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 125L, R 130L, R 130K, R 130L/S 131A, R 158K and R 158L 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 130L and R 130L/S 131A mutants suggested that Arg 130 mutation disrupted the hydrogen bond between Ser 131 and Cys 124. Furthermore, the arsenate binding affinity for R 125L, R 130L and R 158L 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.