Catalytic mechanism of F1Fo-ATP synthase

F1Fo-ATP合酶的催化机制

基本信息

批准号：
8052061
负责人：
JOACHIM WEBER
金额：
$ 10.76万
依托单位：
TEXAS TECH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-28 至 2012-03-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): F1F0-ATP synthase is responsible for the bulk of ATP synthesis in most organisms. It uses a transmembrane proton gradient to synthesize ATP from ADP and Pi, and it hydrolyses ATP to transport protons across the membrane. Both functions are tightly coupled by a unique mechanism, subunit rotation, making ATP synthase a very efficient rotary nanomotor. The broad, long-term goal of this research is to understand the mechanism of ATP synthase in molecular detail. The Specific Aims focus on the connection between substrate binding/turnover in the three catalytic sites and subunit rotation, trying to exploit the available structural information to the fullest. The Specific Aims are: (1) Identification of the high-affinity site, which is responsible for catalysis, in the x-ray structure. Fluorescence resonance energy transfer between tryptophan residues in the rotor and nucleotide analogs will be used to achieve this aim. (2) Investigation of the functional significance of subunit/subunit contacts specific to one of the three catalytic Beta/Alpha interfaces. The functional consequences of (a) preventing these contacts and (b) making them permanent by crosslinking will be tested. (3) Kinetic analysis of nucleotide binding to the catalytic site. ATP binding contributes to driving subunit rotation; the sequence of events leading from the initial contact to closure of the site will be analyzed using tryptophan fluorescence and rapid kinetics. (4) Analysis of the role of the C-terminal domain of Beta in driving rotation. A loop in this domain is generally regarded as instrumental for coupling catalysis to rotation. This hypothesis will be tested by reducing the size of the loop and measurement of the rotational torque produced by ATP hydrolysis. (5) Investigation of the contribution of the Alpha subunit in driving subunit rotation. The role of a loop in the C-terminus of Alpha in transmitting conformational changes between the catalytic sites and the rotor will be analyzed, again using deletions and torque measurements. ATP synthase from E. coli will be used as model in these studies. However, the basic mechanism of all ATP synthases is the same, independent of the source. Mutations in human mitochondrial ATP synthase and related ATP-driven pumps are responsible for a number of diseases.

描述（由申请人提供）：F1F0-ATP 合酶负责大多数生物体中的大部分 ATP 合成。它利用跨膜质子梯度从 ADP 和 Pi 合成 ATP，并水解 ATP 以跨膜运输质子。这两种功能通过一种独特的机制（亚基旋转）紧密耦合，使 ATP 合酶成为非常高效的旋转纳米马达。这项研究的广泛、长期目标是从分子细节上了解 ATP 合酶的机制。具体目标侧重于三个催化位点的底物结合/周转与亚基旋转之间的联系，试图充分利用可用的结构信息。具体目标是： (1) 鉴定 X 射线结构中负责催化的高亲和力位点。转子中色氨酸残基和核苷酸类似物之间的荧光共振能量转移将用于实现这一目标。 (2) 研究三个催化 Beta/Alpha 界面之一特有的亚基/亚基接触的功能意义。将测试（a）防止这些接触和（b）通过交联使其永久化的功能后果。 (3)与催化位点结合的核苷酸的动力学分析。 ATP 结合有助于驱动亚基旋转；将使用色氨酸荧光和快速动力学分析从最初接触到站点关闭的事件顺序。 (4)Beta C端结构域驱动旋转的作用分析。该域中的环通常被认为有助于催化与旋转的耦合。该假设将通过减小环路的尺寸并测量 ATP 水解产生的旋转扭矩来检验。 (5)研究Alpha亚基在驱动亚基旋转中的贡献。将再次使用缺失和扭矩测量来分析 Alpha C 末端环在催化位点和转子之间传递构象变化中的作用。来自大肠杆菌的 ATP 合酶将在这些研究中用作模型。然而，所有 ATP 合酶的基本机制都是相同的，与来源无关。人类线粒体 ATP 合成酶和相关 ATP 驱动泵的突变是导致许多疾病的原因。