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Molecular structure of H^+ transporting ATPase and its rotation mechanisms in the catalysis

H^转运ATP酶的分子结构及其催化旋转机制

基本信息

批准号：
09680622
负责人：
KANAZAWA Hiroshi
金额：
$ 2.05万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Concentrations of various ions including H^+ and Na^+ are strictly regulated within living cells. These regulations are important to keep internal environment of cells and also their growth , which are related to several diseases like cancer, when they are deregulated. Ion transporting proteins in various biological membranes have very important roles in such regulations. Further, some of them have essential roles in energy transduction such as ATP synthesis. In the present study, we have studied structure, function, and regulation for two of these proteins, ATP synthase with techniques of genetic engineering and biochemistry.ATP synthase of Escherichia coli has 8 independent subunits and the genes of subunits were cloned and sequenced. Electrochemical proton gradient across biological membranes and H^+ flow through this membrane bound enzyme is essential for catalysis of ATP synthesis. However, relationship bewteen such complex subunit structure and the catalytic mechanisms of the enzyme is not well understood. Topological arrangement of the subunits should be understood to realize the function of subunits. Here, we developed a new approach with a combination of genetics and biochemistry. We have shown that a new genetic approach, the yeast two-hybrid system, could be applicable to reveal subunit interactions within the enzyme. We have shown tight interactions of alpha-beta gamma-epsilon and b-delta by this procedure. These interactions were further. evidenced by in vitro reconstitution of the subunit complex. Functional importance of the interactions was analyzed by introducing various functionally defective mutations for the interactions. One of the most important findings was b-delta interaction which contributed for the interaction of membrane integral and peripheral portions.

在活细胞中，包括H^+和Na^+在内的各种离子的浓度受到严格的调节。当细胞放松调节时，这些调节对于保持细胞的内部环境及其生长非常重要，这与癌症等多种疾病有关。各种生物膜上的离子转运蛋白在这种调控中起着非常重要的作用。此外，它们中的一些在能量转导如ATP合成中具有重要作用。本研究利用基因工程和生物化学技术对其中的两个蛋白质--ATP合成酶的结构、功能和调控进行了研究，大肠杆菌ATP合成酶有8个独立的亚基，并对亚基基因进行了克隆和测序。生物膜上的电化学质子梯度和H^+流经这种膜结合酶对于催化ATP合成是必不可少的。然而，这种复杂的亚基结构与酶的催化机制之间的关系还不清楚。子单元的功能实现需要理解子单元的拓扑结构。在这里，我们开发了一种结合遗传学和生物化学的新方法。我们已经表明，一种新的遗传方法，酵母双杂交系统，可以适用于揭示酶内的亚基相互作用。我们已经通过这个过程证明了α-β-γ-δ和b-δ的紧密相互作用。这些互动更进一步。通过亚基复合物的体外重建证明。通过引入相互作用的各种功能缺陷突变来分析相互作用的功能重要性。最重要的发现之一是b-δ相互作用，其有助于膜积分和外周部分的相互作用。