PARTICIPATION OF METALS IN THE F1-ATPASE MECHANISM

描述：F1 F0-ATP合成酶利用通过以下途径获得的能量： 代谢物的氧化为大多数重要器官和 组织系统。 对编码F1 F0 ATP合酶的基因的损伤， 由于老化或通过自由基与神经肌肉 虚弱、共济失调和色素性视网膜炎。 破坏程度的增加 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F1 F0 ATP合酶 维持ATP与ADP/磷酸盐的比率远离平衡。 以来 这种酶的催化位点迅速将ATP与 ADP/磷酸盐，使得结合底物的平衡常数和 产物是近似统一的，酶的作用机制是 选择性地释放ATP以维持非平衡状态， 一个未回答的大问题 我们的研究与VO+2探测金属结合 F1-ATP酶的位点开辟了一条研究 这一重要酶的作用机制。 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， 按照配体插入和从配体中置换的顺序， 催化金属中心，我们可以解开机制，使酶 相对于ADP选择性地释放ATP，即使是在浓度梯度下。 首先，在催化位点作为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。 突变体中EPR观察到的变化 酶将通过与VO+2-模型复合物的直接比较来预测， 已知晶体结构含有与 野生型或突变型酶。 第二，在不同的影响， 突变酶结合金属-核苷酸复合物的能力将 与其催化活性相比。 第三，变异人的能力 酶在两种形式的催化位点之间相互转化， 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。 通过将 从我们的EPR研究突变体的晶体结构信息 酶的结构，我们将测量酶插入的能力 并取代金属配体，从而阐明 金属连接和酶促机制之间的变化。

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.