STRUCTURE AND MECHANISM OF F0F1-ATP SYNTHESES

F0F1-ATP 合成的结构和机制

• 批准号: 6179330
• 负责人: RICHARD L CROSS
• 金额: $ 31.76万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-05-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6179330
• 关键词: Escherichia coli; active sites; adenosine diphosphate; adenosine triphosphate; bacterial proteins; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate analog; hydrogen transporting ATP synthase; mitochondrial membrane; nucleotide metabolism; oxidative phosphorylation; polymerase chain reaction; protein sequence; site directed mutagenesis; suppressor mutations

A major energy conversion process in living organisms involves coupling the burning of reduced fuels by respiratory chains to the synthesis of ATP by F1F0 ATP synthases. The enzymes that catalyze these reactions are found embedded in the membranes of mitochondria, chloroplasts, and bacteria, and a transmembrane electrochemical proton gradient appears to function as a required intermediate. The F0 sector of the synthase is composed of membrane spanning subunits (a B2 c12 in E. coli) which conduct protons across the membrane, whereas the F1 sector (alpha3 beta3 gamma delta epsilon) is an extrinsic complex that contains the catalytic sites for ATP synthesis. It has been proposed that proton transport is coupled to ATP synthesis by the rotation of a complex of subunits (gamma epsilon c12) that extends through F0F1. Characterizing this subunit rotation and elucidating other aspects of the mechanism of the synthase are the goals of this proposal. The existence of subunit rotation in F1 is well established but the details of rotational coupling are not well defined. In the proposed studies, a spectral assay will be developed to monitor subunit rotation in fully-active, soluble F1. The approach involves positioning a single fluorescent reporter group on the gamma subunit and a quencher on two of the three catalytic subunits (beta) such that rotation of gamma relative to beta will bring the fluorescent group close to the quenchers to cause transient quenching. This assay will be used to test the kinetic competency of subunit rotation as a required step in ATP synthesis that is coupled to subunit rotation. In contract to F1, subunit rotation in F0 has not been demonstrated. For this purpose, an intersubunit disulfide crosslinking approach will be developed which involves substituting a cys on a c subunit in the c12 oligomer. The test for subunit rotation will be to determine whether the c subunit which is initially in the proper orientation to form a crosslink to subunit a is the same one that occupies that position after catalytic turnover, or whether subunit rotation has moved a different c subunit into that position.

生物体中的主要能量转换过程涉及耦合 通过呼吸链燃烧减少燃料的合成 F1F0 ATP合酶的ATP。催化这些反应的酶是 发现嵌入了线粒体，叶绿体和 细菌和跨膜电化学质子梯度出现 充当所需的中间体。 合酶的F0扇区 由跨膜的亚基（大肠杆菌中的B2 C12）组成 在整个膜上进行质子，而F1扇区（alpha3 beta3 伽玛三角洲epsilon）是一种外在复合物，含有催化 ATP合成的站点。 已经提出质子运输是 通过旋转亚基的旋转（伽玛）旋转与ATP合成 EPSILON C12）延伸到F0F1。 表征这个亚基 旋转并阐明合酶机制的其他方面 是该提议的目标。 F1中的亚基旋转的存在已很好，但 旋转耦合的详细信息没有很好地定义。 在提议中 研究，将开发光谱测定以监视亚基旋转 在完全活跃的可溶性F1中。 该方法涉及定位一个 伽马亚基上的荧光记者组和两个 在三个催化亚基（β）中，伽马旋转 相对于β将使荧光组接近淬火器 引起瞬态淬火。 该测定将用于测试 亚基旋转的动力学能力作为ATP的必需步骤 耦合与亚基旋转的合成。 在与F1的合同中，尚未证明F0中的亚基旋转。 为此，一种亚基间的二硫键交联方法将 开发涉及在c12中替换cys的cys 低聚物。 亚基旋转的测试将是确定是否 最初以适当的方向形成A的C亚基 交叉链接到亚基A是相同的位置 催化周转，或亚基旋转是否移动了不同 c亚基进入该位置。