CHARACTERIZATION OF THE F1-SECTOR OF E COLI H+-ATPASE

大肠杆菌的质子-ATPase负责产生 跨膜质子梯度与氧化合成三磷酸腺苷 磷酸化。这项建议旨在研究结构和催化 聚焦于三个亚基的酶F1区的作用机制 (Alpha、Beta和Gamma)，它们共同构成最小组件 (Alpha3Beta3Gamma)催化活性所需。 对Alpha、Beta和Gamma亚基的修饰将由 定位诱变、定点突变和亲和标记 为了评估特定残基和结构域的重要性 结构和催化中的亚基。函数相关性将是 通过研究修饰对负值的影响而建立的 核苷酸结合、总核苷酸结合、单核苷酸结合的协作性 催化、正催化位协同作用和亚基间 构象相互作用。 为了进一步研究亚基间的相互作用，特别是 对可能涉及催化协同性的接触区域的引用， 部分回复突变株将被分离、鉴定和交联化 实验将在突变的酶上进行。 含有一个或两个突变型Alpha(或Beta)拷贝的杂交酶 将构建亚基和两个或一个拷贝的阿尔法(或贝塔)，以及 确定了它们的催化性能。这样的实验可能会提供一种 F1催化循环三点反应方案的试验 是由我们实验室和其他工作人员提出的。催化作用 将建立正常的Alpha3Beta3Gamma最小单元的性质， 和单独添加纯Delta和Epsilon亚基的效果 将被评估以显示这些亚单位如何影响 F1的整体属性。分离的阿尔法亚单位将被结晶。 离子转运ATPase的能量偶联和催化的本质是 生物学的一个基本问题。这项提议的长期目标是 以推断出对这个问题的分子洞察力。

The proton-ATPase of E. coli is responsible for generation of trans-membrane proton gradients and for ATP synthesis in oxidative phosphorylation. This proposal aims to study structure and catalytic mechanism of the F1-sector of the enzyme by focussing on the three subunits (Alpha, Beta and Gamma) which together form the minimal assembly (Alpha3Beta3Gamma) required for catalytic activity. Modification of Alpha, Beta and Gamma subunits will be carried out by localized mutagenesis, site-directed mutagenesis and affinity labeling in order to assess the importance of specific residues and domains of the subunits in structure and catalysis. Functional correlations will be established by studying the effects of modifications on negative cooperativity of nucleotide binding, total nucleotide binding, single-site catalysis, positive catalytic site cooperativity, and intersubunit conformational interaction. In order to further study intersubunit interactions, and with particular reference to contact regions possibly involved in catalytic cooperativity, partial revertants will be isolated and characterized and cross-linking experiments will be performed on mutant enzymes. Hybrid enzymes containing one or two copies of mutant Alpha (or Beta) subunits and two or one copies of Alpha (or Beta) will be constructed, and their catalytic properties established. Such experiments may provide a test of the cyclical three-site reaction schemes for F1 catalysis which have been proposed by our laboratory and by other workers. Catalytic properties of the normal Alpha3Beta3Gamma minimal unit will be established, and the effects of addition of pure Delta and Epsilon subunits singly and in combination will be assessed to show how these subunits affect the overall properties of F1. Isolated Alpha subunit will be crystallized. The nature of energy-coupling and catalysis in ion-transporting ATPases is a fundamental problem of biology. The long-term goal of this proposal is to deduce molecular insights into this problem.