PARTICIPATION OF METALS IN THE F1-ATPASE MECHANISM

金属参与 F1-ATP 酶机制

• 批准号: 2459508
• 负责人: WAYNE D FRASCH
• 金额: $ 17.35万
• 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 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2459508
• 关键词: Chlamydomonas; active sites; adenosine diphosphate; conformation; electron spin resonance spectroscopy; electronic spectra; enzyme activity; enzyme complex; enzyme mechanism; enzyme model; hydrogen transporting ATP synthase; ligands; mutant; site directed mutagenesis; thermodynamics; vanadium

DESCRIPTION: The F1F0-ATP synthase harnesses the energy obtained by oxidation of metabolites to provide energy for most all vital organ and tissue systems. Damage to the genes that code for the F1F0 ATP synthase as a result of aging or via free radicals are associated with neurologic muscle weakness, ataxia, and retinitis pigmentosa. Increased levels of damage have been found in patients with Parkinson's disease and cardiomyopathies. ATP can serve as the energy currency for a cell because the F1F0 ATP synthase maintains the ratio of ATP to ADP/phosphate away from equilibrium. Since the catalytic sites of this enzyme rapidly interconvert ATP with ADP/phosphate such that the equilibrium constant of the bound substrates and products is approximately unity, the mechanism whereby the enzyme selectively releases ATP to maintain the nonequilibrium condition remains a major unanswered question. Our studies with VO+2 to probe the metal binding sites of the F1-ATPase have opened a fertile new avenue of inquiry into the mechanism of this important enzyme. These studies indicated the ligands change during catalysis. These results led us to the view that, by following the sequence in which ligands are inserted and displaced from the catalytic metal center, we can unravel the mechanism that enables the enzyme to release ATP selectively over ADP, even against a concentration gradient. First, specific groups that serve as metal-ligands at the catalytic site will be identified by observing diagnostic changes in the CW-EPR and/or ESEEM spectra of the metal VO+2 bound to the enzyme that has been altered using site-directed mutagenesis. Changes observed by EPR in the mutant enzyme will be anticipated by direct comparison to VO+2-model complexes of known crystallographic structure that contain ligands comparable to either wild type or mutant enzyme. Second, the effects of the differences in the ability of the mutant enzymes to bind the metal-nucleotide complexes will be compared to their catalytic activity. Third, the ability of the mutant enzymes to interconvert between two forms of the catalytic site that contain the metal-nucleotide complex will allow us to determine the importance of each amino acid in the ability to make this switch. By relating the structural information from our EPR studies of the mutants to the crystal structure of the enzyme, we will measure the ability of the enzyme to insert and displace metal ligands, and we will thereby elucidate the relationship between changes in the metal ligation and the enzymatic mechanism.

描述：F1F0-ATP 合酶利用获得的能量 代谢物的氧化为大多数重要器官提供能量 组织系统。 编码 F1F0 ATP 合酶的基因受损 衰老或自由基的结果与神经肌肉有关 无力、共济失调和色素性视网膜炎。 损害程度增加 已在帕金森病和心肌病患者中发现。 ATP 可以作为细胞的能量货币，因为 F1F0 ATP 合酶 维持 ATP 与 ADP/磷酸盐的比例远离平衡。 自从 该酶的催化位点可快速将 ATP 与 ADP/磷酸盐使得结合底物的平衡常数和 产品大致是统一的，酶的机制 选择性释放 ATP 以维持非平衡状态 主要未回答的问题。 我们用 VO+2 来探测金属结合的研究 F1-ATP酶位点开辟了一条丰富的新途径来探究 这种重要酶的作用机制。 这些研究表明配体 催化过程中发生变化。 这些结果使我们认为，通过 遵循配体插入和替换的顺序 催化金属中心，我们可以解开使酶发挥作用的机制 即使存在浓度梯度，也能相对于 ADP 选择性地释放 ATP。 首先，在催化位点充当金属配体的特定基团 将通过观察 CW-EPR 中的诊断变化和/或 与已改变的酶结合的金属 VO+2 的 ESEEM 光谱 使用定点诱变。 EPR 在突变体中观察到的变化 通过与 VO+2 模型复合物的直接比较可以预测酶 已知的晶体结构包含与任一可比较的配体 野生型或突变型酶。 二、差异的影响 突变酶结合金属-核苷酸复合物的能力将是 与其催化活性相比。 三、变种人的能力 酶在两种形式的催化位点之间相互转化，其中包含 金属-核苷酸复合物将使我们能够确定 每个氨基酸都有能力进行这种转换。 通过关联 来自我们对突变体的 EPR 研究的晶体结构信息 酶的结构，我们将测量酶插入的能力 并取代金属配体，我们将从而阐明这种关系 金属连接和酶促机制的变化之间的关系。