Mechanism of Coupling ATP hydrolysis to Ca^<2+> transport in sarcoplasmic reticulum Ca^<2+>-ATPase

1.肌浆网Ca^（2+）-ATP酶的Mg/F/E（酶）复合物是磷酸化中间体（EP）的稳定类似物。我们已经发现，增溶和脱脂的Mg/F/E对变性非常稳定。2. Ca^<2+>-ATP酶有3个胞质结构域（A、P、N）。我们发现<200>A结构域的瓦尔环与P结构域的相互作用对Ca^&lt;2+&gt;-转运起重要作用。我们已经发现，该环的足够长度对于EP的构象转变是重要的，而EP的构象转变对于Ca^&lt;2+&gt;转运是至关重要的。我们发现在Darier病家系中报道的这个环区的一些突变也会导致EP转换的抑制。4.发现Arg^<334>在EP转换中起重要作用，Tyr^<122>在EP转换后Ca^2+转运中起重要作用。我们提出了一个耦合模型，其中ATP水解的能量通过胞质结构域的大运动传递到Ca^2+转运位点。5.我们证明了Be/F/E、Al/F/E和Mg/F/E配合物是EP水解的基态、过渡态和酶/产物的稳定类似物。我们还发现EP水解过程中催化位点的结构变化将关闭Ca^2+转运的腔门，阻止Ca^2+从腔中漏出。

1.Mg/F/E(Enzyme) complex of sarcoplasmic reticulum Ca^<2+>-ATPase is an stable analog of phosphorylated intermediate(EP). We have found that the solubilized and delipidated Mg/F/E is remarkably stable against denaturation. 2.The Ca^<2+>-ATPase has 3 cytoplasmic domains (A,P,N). We have found that interaction between Val^<200> loop of A domain and P domain is important for Ca^<2+>-translocation from EP.3.Glu40-Ser48 loop connect A domain and the 1st transmembrane segment. We have found that the adequate length of this loop is important for conformational transition of EP, which is critical for Ca^<2+> translocation. We found that some mutations in this loop region reported in the Darier's disease pedigrees will also cause inhibition of EP transition. 4.We have found that Arg^<334> is important for EP transition and Tyr^<122> for Ca^<2+> translocation after the transition of EP. We have proposed a model for coupling, in which energy of ATP hydrolysis is transmitted to Ca^<2+> transport site through the large movements of cytoplasmic domains. 5.We have shown that the complexes Be/F/E,Al/F/E, and Mg/F/E are stable analogs for the ground state, transition state, and enzyme/product in EP hydrolysis. We also found that the structural changes in the catalytic site in EP hydrolysis will close the lumenal gate for Ca^<2+> translocation to prevent Ca^<2+> leak form the lumen.