F1-ATPase Chemical-Mechanical Coupling Mechanisms

F1Fo ATP合成酶是一种多亚基、膜结合的酶，催化合成大多数 人类线粒体酶中几个亚基的突变具有重要的临床意义。这个 酶起到分子马达的作用，驱动某些亚基的旋转运动。这些酶来自于 细菌的线粒体、叶绿体和质膜非常相似。在大肠杆菌中，F1由一个 (Ap)围绕y亚基的3环，也包含5和e亚基。该膜包埋Fo 连接有A亚基和两个B亚基的C10亚基环。在ATP合成过程中， 通过Fo的质子驱动连接了y和esubunit的c10亚单位环的旋转，从而迫使 (cc|3)3环的连续构象变化并导致由三个(Ap)3中的每一个合成ATP 杂二聚体。ATP的水解也可以驱动y亚基朝着相反的方向旋转。当F1 分子被固定在显微镜盖上，在显微镜下可以观察到的探针被固定在 当加入三磷酸腺苷时，可以观察到y亚基，单分子发生旋转。我们的长期目标是澄清 ATPase驱动y亚基旋转的机制。我们提出了诱导机制作为一种工作 将进行严格检验的旋转假说。这一假说假设库仑势起源于 在(a(3)3环和y亚基之间形成氢键和盐桥的残基有助于 Y亚单位扭矩的生成。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以下突变的影响来检验 消除这些相互作用对使用创新的单分子分析方法测量的y亚基旋转速率的影响 我们是为此目的开发的。(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 将被确定包括在y亚基N和C末端的接触。(3)(AP)3环对旋转的贡献 不直接与y亚基相互作用的残基将被检测，包括那些在y亚基之间传递的残基。 亚基和催化部位。(4)(Ap)3环-y亚基相互作用对y亚基旋转的贡献 在已知的F1晶体结构中被识别，但通过跟踪带电的和极性的y的旋转路径来识别 亚基残基位于(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.