F1-ATPase Chemical-Mechanical Coupling Mechanisms

F1 Fo ATP合酶是一种多亚基、膜结合酶，催化大多数ATP的合成。 人线粒体酶中几个亚基的突变具有重要的临床相关性。的 酶就像一个分子马达，驱动一些亚基的旋转运动。酶从 线粒体、叶绿体和细菌的质膜非常相似。在大肠大肠杆菌中，F1由一个 (ap)3环，围绕y亚基，也包含5和e亚基。包埋Fo的膜由以下组成： a亚基和两个B亚基连接到c10亚基环上。在ATP合成过程中， 质子通过Fo驱动c10亚基环的旋转，y和es亚基连接到c10亚基环， 连续的构象变化（CC| 3）3环，并导致从三个（ap）3环中的每一个合成ATP 异二聚体。ATP的水解也可以驱动y亚基向相反方向旋转。当F1 将分子附着在显微镜盖玻片上，并将可以在显微镜下观察的探针附着在 可以观察到γ亚基单分子在加入ATP时旋转。我们的长期目标是阐明 ATP酶驱动y亚基旋转的机制。我们建议将诱导机制作为一种工作机制， 旋转的假设，将严格审查。这一假设假定，库仑电位起源于 在α（3）3环和γ亚基之间形成氢键和盐桥的残基有助于 y子单元扭矩的产生。我们将通过实现以下目标来检验这一假设。(1)P的贡献 Catch Loop-y亚基与y亚基旋转的相互作用将通过测量突变的影响来检查， 消除这些相互作用对Y亚基旋转速率的影响， 我们为此开发的。(2)其他已知的（a（J）3环-y亚基相互作用对y亚基旋转的贡献 将确定包括在y亚基N和C末端的接触。(3)（AP）_3环对转动的贡献 将检查不与y亚基直接相互作用的残基，包括在y亚基之间通讯的残基。 亚基和催化位点。(4)对y亚基旋转的贡献是（ap）3环-y亚基相互作用， 在F1的已知晶体结构中被识别，但通过跟踪带电和极性y的旋转路径来识别 亚基残基围绕（AP）3环的内侧。

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 (ap)3 ring that surrounds the y subunit, and also contains 5 and e 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 y and esubunits are attached that forces sequential conformational changes in the (cc|3)3 ring and results in the synthesis of ATP from each of the three (ap)3 heterodimers. The hydrolysis of ATP can also drive the rotation of the y 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 y 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 y 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 (a(3)3 ring and the y subunit contribute to the generation of y subunit torque. We will test this hypothesis by accomplishing the following aims. (1) The contribution of p Catch Loop-y Subunit interactions to y subunit rotation will be examined by measuring the effects of mutations that eliminate these interactions on the rate of y subunit rotation measured using an innovative single molecule assay that we developed for this purpose. (2) The contribution of other known (a(J)3 ring-y subunit interactions to y subunit rotation will be determined that include contacts at the y subunit N and C termini.(3) The contribution to rotation of (ap)3 ring residues that do not interact directly with the y subunit will be examined including those that communicate between the y subunit and the catalytic sites. (4) The contribution to y subunit rotation of (ap)3 ring-y 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 y subunit residues around the inside of the (ap)3 ring.