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

E.大肠杆菌负责产生 跨膜质子梯度和ATP的氧化合成 磷酸化 该提案旨在研究结构和催化 通过关注三个亚基来研究酶的F1部分的机制 （Alpha、Beta和Gamma），它们共同构成最小集合 （α 3 β 3 γ）催化活性所需。 α、β和γ亚基的修饰将通过 局部突变、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， 结构和催化的亚基。 功能相关性将是 通过研究修改对负面影响的影响， 核苷酸结合协同性，总核苷酸结合，单位点 催化、正催化位点协同性和亚基间 构象相互作用 为了进一步研究亚基间的相互作用， 关于可能涉及催化协同性的接触区域， 部分回复突变体将被分离和表征， 将对突变酶进行实验。 含有一个或两个突变体α（或β）拷贝的杂合酶 亚基和两个或一个拷贝的α（或β）将被构建， 它们的催化性能建立。 这些实验可以提供一个 用于F1催化的循环三中心反应方案的测试， 已经被我们的实验室和其他工作者提出。 催化 将建立正常α 3 β 3 γ最小单元的性质， 以及单独加入纯δ和ε亚基的影响， 将评估这些亚单位如何影响 F1的整体性能。 分离的α亚基将结晶。 离子转运ATP酶中能量耦合和催化的本质是 生物学的基本问题。 该提案的长期目标是 来推导出这个问题的分子见解。

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.