Catalytic mechanism of F1Fo-ATP synthase

F1Fo-ATP合酶的催化机制

• 批准号: 7116433
• 负责人: JOACHIM WEBER
• 金额: $ 22.68万
• 依托单位: TEXAS TECH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7116433
• 关键词: X ray crystallography; active sites; chemical kinetics; enzyme activity; enzyme structure; fluorescence spectrometry; gene mutation; hydrogen transporting ATP synthase; hydrolysis; molecular dynamics; protein structure function; site directed mutagenesis; structural biology; X ray crystallography; active sites; chemical kinetics; enzyme activity; enzyme structure; fluorescence spectrometry; gene mutation; hydrogen transporting ATP synthase; hydrolysis; molecular dynamics; protein structure function; site directed mutagenesis; structural biology

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的大部分ATP合成。它使用跨膜质子梯度从ADP和PI合成ATP，并水解ATP以在整个膜上运输质子。这两个功能都通过独特的机制（亚基旋转）紧密耦合，使ATP合酶成为非常有效的旋转纳米能力。这项研究的广泛长期目标是从分子细节中了解ATP合酶的机制。 该特定目的集中于三个催化位点和亚基旋转中的底物结合/周转之间的连接，试图充分利用可用的结构信息。具体目的是：（1）X射线结构中负责催化的高亲和力位点的识别。转子和核苷酸类似物中的色氨酸残基之间的荧光共振能量转移将用于实现此目标。 （2）研究特定于三个催化β/α界面之一的亚基/亚基接触的功能意义。 （a）防止这些接触的功能后果以及（b）将通过交联使它们永久进行测试。 （3）核苷酸与催化位点结合的动力学分析。 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初始接触到位点关闭的事件序列。 （4）分析β的C末端结构域在驱动旋转中的作用。该域中的循环通常被认为是将催化与旋转耦合的工具。该假设将通过减少环路的大小和ATP水解产生的旋转扭矩的测量来检验。 （5）研究α亚基在驱动亚基旋转方面的贡献。循环在α的C端在催化位点和转子之间传输构象变化中的作用将再次使用缺失和扭矩测量结果进行分析。 来自大肠杆菌的ATP合酶将在这些研究中用作模型。但是，所有ATP合酶的基本机制都是相同的，与来源无关。人线粒体ATP合酶和相关ATP驱动泵的突变导致多种疾病。