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

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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.