Structure and Catalytic Mechanism of Ion-translocating ATPases

离子转位ATP酶转运离子例如H 2 、Na 2 、K 2 和Ca 2 ，并与ATP的水解偶联。 ATP结合（催化）位点的详细结构、ATP水解与离子传输耦合的机制以及离子传输途径的机制和结构在分子水平上仍然未知。在本项目中，我们研究了主要来自大肠杆菌的 H^ -ATPase (F_1F_O) 以及来自猪和人类的 H^ /K^ ATPase。H^ -ATPase 由 F_1 扇区（α、β、γ、δ 和 epsilon 亚基）和 F_O（a__-、b__- 和 c__- 亚基）形成。我们通过随机和定点诱变的组合方法分析了 ATP 结合位点和 H^ 途径。 ATP 结合位点位于 β 亚基的 149 - 156 个残基和 Tyr-285 残基（从氨基末端编号）附近。 Lys-155 紧邻 aTP 的 γ 磷酰基部分。显示了 γ 亚基的 Gln-269 和 Thr-277 之间的残基对于 ATP 酶活性的重要性。大约一半的ε亚基被证明是可有可无的。我们使用类似的方法分析了H^途径(F<@2O<@D2)：H<@D1@>D1易位的必需氨基酸残基之一位于距离a<@D5-@>D5亚基的羧基末端20个残基内。 b D5-D5 亚基羧基末端的两个残基对于F D2O D2 的组装是必需的。我们克隆了H 2 /K 3 ATP酶的cDNA（猪）和基因组DNA（人）并确定了它们的核苷酸序列。 H + /K + ATP酶的氨基酸序列与其他离子转位ATP酶例如Na + /K + -和Ca + 2 -ATP酶密切同源。我们还表明，磷酸吡哆醛与 H+ /K+ ATP 酶的 Lys-497 结合，并且该赖氨酸残基形成 ATP 结合位点。我们提出了酶的催化位点和 H^ 路径的模型。

Ion-translocating atpases transport ions such as H^+, Na^+, K^+ and Ca^<2+> coupling with hydrolysis of ATP. Detailed structure of the ATP binding (catalytic) site, mechanism of hydrolysis of ATP coupling with ion-transport and mechanism and structure of the ion-transport pathway are still unknown in molecular level. In this project we studied H^+-ATPase (F_1F_O) mainly from Escherichia coli and H^+/K^+ ATPase from hog and human.H^+-ATPase is formed from F_1 sector (alpha, beta, gamma, delta and epsilon subunits) and F_O (a__-, b__- and c__- subunits). We analyzed ATP binding site(s) and H^+ pathway by conbined approach of random and site-directed mutagenesis. ATP binding site was located near 149 - 156 residues and Tyr-285 residue (numbered from the amino-terminus) of the beta subunit. Lys-155 was located closely near the gamma phosphoryl moiety of aTP. Importance of residues between Gln-269 and Thr-277 of the gamma subunit for ATPase activity was shown. About half of the epsilon subunit was shown to be dispensible. We analyzed H^+ pathway (F<@2O<@D2) using the similar approach: one of the essential amino acid residues for H<@D1+@>D1 translocation was located within 20 residues from the carboxyl terminus of the a<@D5-@>D5 subunit. Two residues from the carboxyl-terminus of the b<@D5-@>D5 subunit were essential for assembly of F<@D2O@>D2.We cloned cDNA (pig) and genomic DNA (human) of H^+/K^+ ATPase and determined their nucleotide sequences. The amino acid sequence of H^+/K^+ ATPase was closely homologous to other ion-translocating ATPases such as Na^+/K^+- and Ca^<2+>-ATPase. We also showed that pyridoxal phosphate binds to Lys-497 of the H^+/K^+ ATPase and this lysine residue formed ATP binding site. We proposed a model of catalytic site and H^+ pathay of the enzyme.