PARTICIPATION OF METALS IN THE F1-ATPASE MECHANISM

描述：F1F0-ATP合成酶利用通过 代谢产物的氧化为几乎所有的重要器官提供能量 组织系统。对编码F1F0 ATP合成酶的基因的损伤 衰老的结果或通过自由基与神经肌肉有关 虚弱、共济失调和视网膜色素变性。损害程度的增加已经 在帕金森氏症和心肌病患者中被发现。ATP 可以作为细胞的能量货币，因为F1F0 ATP合成酶 保持ATP与ADP/磷酸盐的比率远离平衡。自.以来 这种酶的催化部位能迅速地将三磷酸腺苷与 ADP/磷酸盐使结合底物的平衡常数和 产物大致是统一的，酶由此产生的机制 选择性地释放三磷酸腺苷以维持非平衡状态 重大悬而未决的问题。我们用VO+2探索金属结合的研究 F1-ATPase的网站开辟了一条肥沃的新途径来调查 这种重要酶的作用机制。这些研究表明，这些配体 在催化过程中发生变化。这些结果使我们得出这样的观点，即 按照配体插入和移位的顺序 催化金属中心，我们可以解开使酶 通过ADP选择性地释放ATP，即使在浓度梯度的情况下也是如此。 首先，在催化部位充当金属配体的特定基团 将通过观察CW-EPR和/或 金属VO+2与酶结合后的ESEEM光谱 使用定点突变。EPR在突变体中观察到的变化 通过直接与VO+2模型络合物进行比较，可以预见酶的存在。 含有可与以下任一种配体相媲美的配体的已知晶体结构 野生型或突变酶。第二，中美关系差异的影响 突变酶结合金属-核苷酸复合体的能力将是 与它们的催化活性相比。第三，突变体的能力 在两种形式的催化部位之间相互转换的酶，其中包含 金属-核苷酸复合体将使我们能够确定 每一种氨基酸都有能力进行这种转换。通过将 从我们的EPR研究到晶体的突变体的结构信息 酶的结构，我们将测量酶的插入能力 并取代金属配体，因此我们将阐明这种关系 在金属连接和酶机制的变化之间。

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.