Research on molecular mechanism of sarcoplasmic reticulum Ca^<2+>-ATPase with site-directed mutagenesis

肌浆蛋白Ca~(2+)&t；~(2+)-ATPase催化Ca~(2+)~(2+)~(2+)转运到ATP水解液中。在催化循环中，三磷酸腺苷的γ-磷酸基团转移到天冬氨酸351上形成磷酸化中间体(EP)，然后EP发生异构化和水解反应。在本研究项目中，我们获得了以下新的发现(a-d)：(A)用SH_D的N-乙基马来酰亚胺(Cys344和Cys364)修饰EP可以抑制EP的异构化。然而，当添加有机溶剂降低介质中的水活度时，发生了异构化反应。结果表明，在EP异构化过程中，SH_D区在清除催化部位的水分子中起着重要作用。(B)该酶的胞质区由磷酸化、ATP结合和A-结构域组成。用定点突变的方法研究了A结构域的可能作用。发现A区的Arg 198对EP的快速水解是必不可少的，在EP的水解过程中可能位于磷酸化部位附近。(C)在EP的水解过程中，A区的His5位于磷酸化部位附近。通过定点突变和脉冲追逐分析该酶蛋白在细胞中的表达和降解，进一步发现His5及其周围区域对ER介导的质量控制非常敏感，因此对酶的正确折叠的三级结构的形成和稳定非常重要。(D)EP异构化时，Ca^&lt；2+和Gt；-ATPase几乎完全抵抗胰酶、蛋白酶K和V8-蛋白酶，因此形成了非常紧密的构象，其中三个细胞质结构域聚集在一起。

Sarcoplasmuc retuculum Ca^<2+> -ATPase catalyzes Ca^<2+> transport coupled to ATP hydrolysus. In the catalytic cycle, γ-phosphate group of ATP is transferred to Asp351 to from phosphorylated intermediate (EP), and isomerization and hydrolysis of EP occurs successively. In this research project, we obtained the following new findings (a-d).(a) The isomerization of EP was inhibited by modification with N-ethylmaleimide of SH_D (Cys344 ＆ Cys364) of the enzyme. However, when the water activity in the medium was reduced by addition of organic solvents, the isomerization was shown to occur. Results indicated that the SH_D-region has important roles in the elimination of water molecules from the catalytic site during the isomerization of EP.(b) The cytoplasmic region of this enzyme consist of phosphorylation-, ATP binding-, and A-domains. The possible role of A-domain was investigated by using site-directed mutagenesis. It was found that Arg 198 in A-domain is essential for the rapid hydrolysis of EP, and is likely located near the phosphorylation site during hydrolysis of EP.(c) His5 in A-domain was found to be located near the phosphorylation site during hydrolysis of EP.Using site-directed mutagenesis and pulse-chase analysis of the expression and degradation of the enzyme protein in cells, it was further found that His5 and the surrounding region are very sensitive to the ER-mediated quality control, and thus important for formation and stabilization of the correctly folded tertiary structure of the enzyme.(d) Upon isomerization of EP, the Ca^<2+>-ATPase becomes almost completely resistant to trypsin, proteinase K, and V8-protease and therefore forms very compact conformation in which the three cytoplasmic domains gather.