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

肌浆重组Ca^<2+> -ATP酶催化Ca^<2+>转运偶联ATP水解。在催化循环中，ATP的γ-磷酸基团被转移到Asp351上，从磷酸化中间体（EP）中分离出来，EP的异构化和水解依次发生。在这个研究项目中，我们获得了以下新发现（a-d）。(a)用n -乙基马来酰亚胺修饰酶的SH_D （Cys344和Cys364）抑制了EP的异构化。然而，当介质中的水活度通过添加有机溶剂而降低时，异构化就会发生。结果表明，在EP异构化过程中，sh_d区在催化位点消除水分子中起重要作用。(b)该酶的细胞质区由磷酸化-、ATP结合-和a结构域组成。利用定点诱变研究了a结构域可能的作用。结果表明，a结构域的Arg 198对于EP的快速水解至关重要，可能位于EP水解过程中的磷酸化位点附近。(c)在EP水解过程中发现a结构域的His5位于磷酸化位点附近。通过定点诱变和脉冲追踪分析细胞中酶蛋白的表达和降解，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，因此对酶正确折叠的三级结构的形成和稳定非常重要。(d) EP异构化后，Ca^<2+>- atp酶几乎完全抵抗胰蛋白酶、蛋白酶K和v8蛋白酶，因此形成非常紧密的构象，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.