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.

包括H^+和Na^+在内的各种离子的浓度在活细胞中受到严格调节。这些法规对于保持细胞的内部环境以及它们的生长很重要，它们的生长与癌症（如癌症）相关时，它们被放松管制。各种生物膜中的离子运输蛋白在此类法规中具有非常重要的作用。此外，其中一些在诸如ATP合成之类的能量转导中具有重要作用。在本研究中，我们研究了其中两种蛋白质的结构，功能和调节，ATP合酶具有基因工程和生物化学技术。大肠杆菌的ATP合酶具有8个独立亚基，并将亚基的基因克隆并进行了测序。跨生物膜的电化学质子梯度和H^+流过这种膜结合酶对于ATP合成的催化至关重要。但是，这种复杂的亚基结构和酶的催化机制的关系尚不清楚。应理解亚基的拓扑布置以实现亚基的功能。在这里，我们开发了一种新方法，结合了遗传学和生物化学。我们已经表明，一种新的遗传方法，即酵母双杂交系统，可用于揭示酶内部的亚基相互作用。通过此过程，我们已经显示出Alpha-Beta Gamma-Epsilon和B-Delta的紧密相互作用。这些相互作用是进一步的。亚基复合物的体外重构证明了这一点。通过引入相互作用的各种功能有缺陷的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-Delta相互作用，这有助于膜积分和外围部分的相互作用。