PARTICIPATION OF METALS IN THE F1-ATPASE MECHANISM

金属参与 F1-ATP 酶机制

• 批准号: 6018977
• 负责人: WAYNE D FRASCH
• 金额: $ 18.76万
• 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 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018977
• 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合酶利用通过 代谢物的氧化以为大多数重要的器官提供能量， 组织系统。 损害为F1F0 ATP合酶编码的基因损坏 衰老或通过自由基的结果与神经系统肌肉有关 无力，共济失调和色素性视网膜炎。 损害水平增加具有 在帕金森氏病和心肌病的患者中发现。 ATP 可以用作电池的能源货币，因为F1F0 ATP合酶 保持ATP与ADP/磷酸盐的比率远离平衡。 自从 该酶的催化位点与 ADP/磷酸盐，使结合底物的平衡常数和 产品大约是统一的，即酶的机制 有选择地释放ATP以维持非平衡状态 主要的未解决问题。 我们对VO+2的研究以探测金属结合 F1-ATPase的地点已经为该地区开辟了新的调查途径 这种重要酶的机制。 这些研究表明配体 催化过程中的变化。 这些结果使我们认为， 遵循插入配体并从中移位的顺序 催化金属中心，我们可以揭开实现酶的机制 在ADP上有选择地释放ATP，甚至针对浓度梯度。 首先，在催化位点充当金属配体的特定组 将通过观察CW-EPR和/或的诊断变化来确定 金属VO+2的ESEEM光谱与已改变的酶结合 使用定向诱变。 EPR在突变体中观察到的变化 通过直接比较与VO+2模型复合物的直接比较，可以预期酶 已知的晶体学结构，包含与任何一个相当的配体 野生型或突变酶。 第二，差异的影响 突变酶结合金属核苷酸络合物的能力将是 与它们的催化活性相比。 第三，突变体的能力 酶在包含的两种形式的催化位点之间互连 金属核苷酸络合物将使我们能够确定 每个氨基酸都具有使此开关的能力。 通过关联 从我们对突变体到晶体的EPR研究的结构信息 酶的结构，我们将测量酶插入的能力 并取代金属配体，我们将阐明这种关系 金属连接的变化与酶促机理之间。