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

ATPase有三种类型：F-ATPase，合成ATP，存在于线粒体、叶绿体和细菌膜中；V-ATPase，在溶酶体和突触小泡等膜上水解ATP并形成电化学质子梯度；P-ATPase，形成磷酸酶中间体，如胃H+/K++ATPase和酵母H^+ATPase。我们从比较的角度对这三种ATPase进行了研究，主要集中在催化部位及其与H~+转位的偶联。特别是对F-ATPase和V-ATPase进行了研究。主要研究结果如下：(1)我们发现F-ATPase的Lys-155和Thr-156是参与底物结合步骤的催化残基：突变的酶(Lys-155*Ala、Thr、Ser或Arg、Thr-156 Ala、Cys、Asp或Ser)不具有ATPase和ATP合成酶活性。这些残基与Lys-155和Thr-156一起在V-ATPase中保守，表明它们也在V-ATPase的催化部位。(3)遗传学研究表明，F-ATPase的伽马亚基调节催化与H~+转运之间的偶联。突变的MET-23*Lys具有野生型ATPase活性，但没有H~(++)转运和ATP合成。引入第二个突变(例如Gln-269*Arg)可恢复H~+转运或ATP合成。这一系列研究表明，氨基和羧基末端区域的相互作用对偶联非常重要。(4)克隆了V-ATPase的催化亚基和质子途径，并形成了与F-ATPase相应的亚基类似的亚基。(5)我们发现突触小泡中含有大量的V-ATPase：约占粘膜膜蛋白的20%。这种酶被证明可以驱动神经递质运输科学研究助学金(B)

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)