Studies on H^+-ATPase:Catlytic site and Coupling between Catalysis

三种类型的ATP酶耦合ATP水解（合成）和H^+转运：F-ATP酶，合成ATP，存在于线粒体、叶绿体和细菌膜中; V-ATP酶，水解ATP，在内膜如溶酶体和突触囊泡中形成电化学质子梯度; P-ATP酶，形成磷酸酶中间体，如胃H^+/K^+ ATP酶和酵母H^+ ATP酶。我们从比较的角度对这三种类型的ATP酶进行了研究，主要集中在催化位点及其与H^+转运的偶联上。重点研究了F-ATP酶和V-ATP酶。主要研究结果如下。(1)我们发现F-ATP酶的Lys-155和Thr-156是参与底物结合步骤的催化残基：突变酶（Lys-155* Ala、Thr、Ser或Arg，Thr-156 Ala、Cys、Asp或Ser）没有ATP酶和ATP合酶活性。(2)突变体及其抑制子的分析表明，β亚基的Gly-172、Ser-174、Glu-192和Gly-149位于F-ATP酶的催化位点附近或催化位点内。这些残基与Lys-155和Thr-156一起在V-ATPase中是保守的，表明它们也在V-ATPase的催化位点中。(3)遗传学研究表明，F-ATP酶的γ亚基调节催化与H^+转运之间的偶联。γ亚基的Met-23* Lys突变体具有野生型ATP酶活性，但没有H^+转运或ATP合成。引入第二个突变（例如，Gln-269* Arg）恢复H^+转运或ATP合成。这一系列的研究表明，氨基和羧基末端区域的相互作用是重要的耦合。(4)V-ATP酶的催化亚基和质子途径被克隆并形成类似于F-ATP酶的相应亚基。(5)我们发现突触囊泡中含有大量的V-ATP酶，约占囊泡膜蛋白的20%。这种酶被证明可以驱动神经递质的运输

Three type ATPase couple ATP hydrolysis(synthesis) and H^+ translocation : F-ATPase, synthesizing ATP and found in mitochondria, chloroplasts and bacterial membranes; V-ATPase, hydrolyzing ATP and forming electrochemical proton gradient in endomembranes such as lysosomes and synaptic vesicles; P-ATPase, forming phosphoenzyme intermediate such as gastric H^+/K^+ ATPase and yeast H^+ ATPase. We are studying these three type ATPase from the comparative aspects, and focussed mainly catalytic site and its coupling with H^+ translocation. Especially F-ATPase and V-ATPase were studied in the research. Major results are summarized as following.(1) We showed Lys-155 and Thr-156 of F-ATPasse are shown to be catalytic residues participating substrate-binding steps : mutant enzymes (Lys-155* Ala, Thr, Ser, or Arg, Thr-156 Ala, Cys, Asp or Ser) had no ATPase and ATP synthase activities.(2) Analysis of mutants and their surpressers indicated that Gly-172, Ser-174, Glu-192 and Gly-149 of the beta subunit are near or in the catalytic site of F-ATPase. These residues together with Lys-155 and Thr-156 are conserved in V-ATPase, suggesting they are also in the catalytic site of V-ATPase.(3) The gamma subunit of F-ATPase was suggested to be regulating coupling between catalysis and H^+ transport by genetic studies. Met-23* Lys mutant of the gamma subunit had wild-type ATPase activity but no H^+ transport or ATP synthesis. Introducing the second mutation (eg. Gln-269* Arg) restored H^+ transport or ATP synthesis. This line of studies indicated that interaction of amino and carboxyl terminal regions are important for the coupling.(4) Catalytic subunit and proton pathway of V-ATPase were cloned and formed similar to the corresponding subunits of F-ATPase.(5) We found that synaptic vesicles contain large amount of V-ATPase : about 20% of the visicle membrane protein. This enzyme was shown to drive neurotransmitter transport Grant-in-Aid for Scientific Research(B)