Structure and Catalytic Mechanism of Ion-translocating ATPases

离子转运ATP酶与ATP水解偶联运输H^+、Na^+、K^+和Ca^<2+>等离子。在分子水平上，ATP结合（催化）位点的详细结构、ATP与离子转运偶联的水解机制以及离子转运途径的机制和结构尚不清楚。本课题研究的H^+- atp酶（F_1F_O）主要来自大肠杆菌，H^+/K^+ atp酶主要来自猪和人，H^+- atp酶主要由F_1区（α、β、γ、δ和epsilon亚基）和F_O （a__-、b__-和c__-亚基）组成。我们采用随机诱变和定点诱变相结合的方法分析了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tyr285个残基（从氨基端开始编号）附近。Lys-155位于aTP的γ磷酸基附近。γ亚基Gln-269和Thr-277之间的残基对atp酶活性的重要性得到了证明。大约一半的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a<@D5-@>D5亚基羧基末端的20个残基内。b<@D5-@>D5亚基羧基末端的两个残基对于F<@ d20 @>D2的组装是必需的。我们克隆了H^+/K^+ 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Na^+/K^+-和Ca^<2+>- atp酶等离子易位atp酶具有密切的同源性。我们还发现磷酸吡哆醛与H^+/K^+ ATP酶的赖氨酸-497结合，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^+途径模型。

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.