Structure and Catalytic Mechanism of Ion-translocating ATPases

离子转运型ATPase是H~(++)、Na~(++)、K~(++)和Ca~(2+)~(2+)等离子的转运体，与ATP水解酶偶联。ATP结合(催化)部位的详细结构、ATP水解与离子转运偶联的机制以及离子转运途径的机制和结构在分子水平上仍不清楚。在本课题中，我们主要研究了来自大肠杆菌的H~+-ATPase(F_1F_O)和来自猪和人的H~+/K~+-ATPase。H~+-ATPase是由F_1扇区(α、β、γ、Delta和epsilon亚基)和F_O(a_-、b_-和c_-亚基)形成的。我们采用随机突变和定点突变相结合的方法对三磷酸腺苷结合位点(S)和H~+途径进行了分析。ATP结合位点位于β亚基149-156个残基和Tyr-285个残基(从氨基末端开始编号)附近。Lys-155位于三磷酸腺苷的γ-磷酸部分附近。谷氨酰胺-269和苏氨酸-277之间的残基对ATPase活性的重要性被证明。大约一半的epsilon亚基被证明是可分配的。我们用类似的方法分析了H~+途径(F&lt；@2O&lt；@D2)：H&lt；@d1+@&gt；D1易位的一个必需氨基酸残基位于a&lt；@D5-@&gt；D5亚基的羧基末端20个残基以内。在B&lt；@D5-@&gt；D5亚基的羧基末端有两个残基是F&lt；@D2O@&gt；D2组装所必需的。我们克隆了H^+/K^+ATPase的cDNAs(猪)和基因组DNA(人)。H^+/K^+ATPase的氨基酸序列与其他离子转运型ATPase如Na+/K^+-和Ca^&lt；2+&Gt；-ATPase有较高的同源性。我们还发现磷酸吡哆醛与H^+/K^+ATPase的Lys-497结合，这个赖氨酸残基形成了ATP结合部位。提出了该酶的催化位置和H~+途径的模型。

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.