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

1.肌浆网Ca~(2+)-ATPase的Mg/F/E(酶)复合体是磷酸化中间体(EP)的稳定类似物。我们发现，增溶和脱脂的Mg/F/E对变性具有显著的稳定性。2.Ca^&lt；2&gt；-ATPase有A、P、N三个胞质结构域。我们发现A结构域和P结构域的Val^&lt；200和P结构域之间的相互作用对EP.3Glu40-Ser48环连接A结构域和第一跨膜片段的Ca^&lt；2&gt；-易位是重要的。我们发现，这个环的适当长度对于EP的构象转变是重要的，而EP的构象转变是Ca^&lt；2&gt；移位的关键。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.我们发现Arg^&lt；334&gt；对EP转换起重要作用，Tyr^&lt；122&gt；在EP转换后对Ca^&lt；2&gt；易位起重要作用。我们提出了一个偶联模型，在该模型中，ATP水解的能量通过细胞质结构域的大运动传递到钙转运部位。5.研究表明，配合物Be/F/E、Al/F/E和Mg/F/E是EP水解酶的基态、过渡态和酶/产物的稳定类似物。我们还发现，EP水解酶催化部位的结构变化将关闭腔内Ca^&lt；2&gt；转位的门，以防止Ca^&lt；2&gt；从腔内泄漏。

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.