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。我们发现溶解和降解的Mg/F/E具有显著的抗变性稳定性。2.Ca^<2+>- atp酶具有3个胞质结构域（A，P，N）。我们发现A域和P域的Val^<200>环之间的相互作用对于Ca^<2+>-从EP.3转运是重要的。Glu40-Ser48环连接A结构域和第一跨膜段。我们发现该环的适当长度对于EP的构象转变非常重要，而EP的构象转变对于Ca^<2+>易位至关重要。我们发现在Darier病谱系中报道的这个环区的一些突变也会导致EP转化的抑制。4.我们发现Arg^<334>对EP跃迁很重要，Tyr^<122>对EP跃迁后Ca^<2+>易位很重要。我们提出了一个耦合模型，其中ATP水解的能量通过细胞质结构域的大运动传递到Ca^<2+>转运位点。5.我们已经证明，配合物Be/F/E、Al/F/E和Mg/F/E是EP水解过程中基态、过渡态和酶/产物的稳定类似物。我们还发现，EP水解催化位点的结构变化将关闭Ca^<2+>转运的管腔门，以防止Ca^<2+>从管腔泄漏。

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.