The principal goal of this project is to determine mechanistic feature of the yeast Saccharomyces cerevisiae mitochondrial ATP synthase. This work is particularly relevant to cardiac tissue which is almost completely dependent on the mitochondrial ATP synthase for aerobic synthesis of ATP. An understanding of the structure and function of the ATP synthase is essential before mitochondrial myopathies and pathologies that alter oxidative-phosphorylation can be completely understood and treated. This study will reveal important basic features of the ATP synthase and, since the yeast enzyme is highly homologous to the human enzyme, it is directly applicable to the understanding and treatment of human diseases. The ATP synthase is composed of a water soluble portion, the F1 ATPase, and a membrane portion, Fo. The recent report of the crystal structure of bovine F1-ATPase at 2.A provides a number of exciting hypothesis that can be tested. First, the structures of the alpha and beta-subunits of F1 are divided into three domains. The top domain is a beta-barrel structure and it is postulated to provide structural support tot he enzyme complex by heterodimer interactions between the alpha and beta-subunits. This hypothesis will be tested by making chimeric enzymes and by biochemical and biophysical studies of the beta-barrel domains. Second, a number of amino acids are located at or near the active site of the enzyme. Roles for two of these residues, beta-Val164 and beta-Glu188, are suggested by the crystal structure and from prior genetic and biochemical studies. beta- Glu188 is suggested to act as the catalytic base in the reaction mechanism. beta-Val164 is at the end of the P-loop motif and is suggested to provide critical interactions with the adenine ring. These hypotheses will be tested by select mutants and by biochemical studies. There are two aims that are not directly based on the crystal structure of the ATPase, but are aided by the structure. First, an aspect of the binding change hypothesis will be tested. Specifically, the hypothesis suggests that only one of the three active sites is engaged in ATP synthesis at any given time.
This aim asks how many functional active sites are required to for an active enzyme? Mutants will be expressed in a wild type background to produce a population of enzyme molecules with zero, one, two and three mutant beta-subunits. The different hybrid molecules will be separated using three unique affinity tags the beta-subunits. After purification, biochemical studies will be used to determine the effect of the number defective active sites on the activity of the enzyme. Finally, the structural interaction of the oligomycin sensitivity conferring protein with F1 ATPase will be determined. OSCP may provide critical links between Fo and F1 and may be critical in the energy transduction pathway. This analysis will combine biochemical and genetic analysis to determine the residues in OSCP that interact with F1 and identify the residues in F1 that interact with OSCP. This project will provide information on the structure and function of the mitochondrial ATP synthase. In addition, this project will expand upon the broad understanding of the mechanism of catalysis.
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