F1-ATPase Chemical-Mechanical Coupling Mechanisms

F1-ATP酶化学机械耦合机制

• 批准号: 7049224
• 负责人: WAYNE D FRASCH
• 金额: $ 27.46万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7049224
• 关键词: Chlamydomonas; active sites; adenosine triphosphate; conformation; electron spin resonance spectroscopy; enzyme activity; enzyme mechanism; fluorescence microscopy; gene mutation; hydrogen transporting ATP synthase; interferometry; liposomes; magnesium ion; molecular dynamics; oxidative phosphorylation; phosphates; protein biosynthesis; site directed mutagenesis; tyrosine

DESCRIPTION (provided by applicant): The F1Fo ATP synthase is a multisubunit, membrane-bound enzyme that catalyzes the synthesis of the majority of cellular ATP. Mutations in several subunits in the human mitochondrial enzyme are of important clinical relevance. The enzyme works as a molecular motor that drives the rotary motion of some of the subunits. The enzymes from mitochondria, chloroplasts and the plasma membranes of bacteria are closely similar. In E. coli, F1 is composed of an (alpha-beta)3 ring that surrounds the gamma subunit, and also contains delta and epsilon subunits. The membrane embedded Fo is composed of a c10 subunit ring to which is attached the a subunit and two b subunits. During ATP synthesis, the movement of protons through Fo drives the rotation of the c10 subunit ring to which the gamma and epsilon subunits are attached that forces sequential conformational changes in the (alpha-beta)3 ring and results in the synthesis of ATP from each of the three (alpha-beta)3 heterodimers. The hydrolysis of ATP can also drive the rotation of the gamma subunit in the opposite direction. When F1 molecules are attached to a microscope cover slip, and a probe that can be observed under a microscope is attached to the gamma subunit, single molecules can be observed to rotate upon addition of ATP. Our long term objective is to elucidate the mechanism of ATPase-driven rotation of the gamma subunit. We propose the induction mechanism as a working hypothesis for rotation that will be critically examined. This hypothesis posits that Coulombic potential originating from residues that form hydrogen bonds and salt bridges between the (alpha-beta)3 ring and the gamma subunit contribute to the generation of gamma subunit torque. We will test this hypothesis by accomplishing the following aims. (1) The contribution of beta Catch Loop-gamma Subunit interactions to gamma subunit rotation will be examined by measuring the effects of mutations that eliminate these interactions on the rate of gamma subunit rotation measured using an innovative single molecule assay that we developed for this purpose. (2) The contribution of other known (alpha-beta)3 ring-gamma subunit interactions to gamma subunit rotation will be determined that include contacts at the gamma subunit N and C termini. (3) The contribution to rotation of (alpha-beta)3 ring residues that do not interact directly with the gamma subunit will be examined including those that communicate between the gamma subunit and the catalytic sites. (4) The contribution to gamma subunit rotation of (alpha-beta)3 ring-gamma subunit interactions that have not been identified in known crystal structures of F1 but are identified by tracking the rotational path of charged and polar gamma subunit residues around the inside of the (alpha-beta)3 ring.

描述（由申请人提供）：F1 Fo ATP合酶是一种多亚基、膜结合酶，催化大部分细胞ATP的合成。人线粒体酶中几个亚基的突变具有重要的临床意义。这种酶就像一个分子马达，驱动一些亚基的旋转运动。来自线粒体、叶绿体和细菌质膜的酶非常相似。在大肠在大肠杆菌中，F1由围绕γ亚基的（α-β）3环组成，并且还包含δ和β亚基。膜包埋的Fo由一个c10亚基环组成，a亚基和两个B亚基连接在该环上。在ATP合成过程中，质子通过Fo的运动驱动c10亚基环的旋转，γ亚基和γ亚基连接到c10亚基环上，这迫使（α-β）3环中的连续构象变化，并导致从三个（α-β）3异二聚体中的每一个合成ATP。ATP的水解也可以驱动γ亚基向相反方向旋转。当F1分子附着在显微镜盖玻片上，并且可以在显微镜下观察到的探针附着在γ亚基上时，可以观察到单个分子在添加ATP时旋转。我们的长期目标是阐明ATP酶驱动的γ亚基旋转的机制。我们提出的诱导机制作为一个工作假设旋转，将严格审查。该假设假定源自在（α-β）3环和γ亚基之间形成氢键和盐桥的残基的库仑电位有助于产生γ亚基扭矩。我们将通过实现以下目标来检验这一假设。(1)β捕获环-γ亚基相互作用对γ亚基旋转的贡献将通过测量消除这些相互作用的突变对γ亚基旋转速率的影响来检查，所述γ亚基旋转速率使用我们为此目的开发的创新单分子测定法来测量。(2)将确定其他已知的（α-β）3环-γ亚基相互作用对γ亚基旋转的贡献，包括γ亚基N和C末端的接触。(3)将检查不与γ亚基直接相互作用的（α-β）3环残基对旋转的贡献，包括在γ亚基和催化位点之间通信的那些残基。(4)（α-β）3环-γ亚基相互作用对γ亚基旋转的贡献，在F1的已知晶体结构中尚未确定，但通过跟踪（α-β）3环内部周围带电和极性γ亚基残基的旋转路径来确定。