The overall goal of this project is to fully elucidate the mechanism of action and cellular role of the enzyme yeast inorganic pyrophosphatase. For the three year period of support requested, the specific aims fall into four areas: I. Studies on native PPase, II. Isolation and sequencing of a yeast PPase clone, III. Generation of point-specific PPase mutants for biochemical studies, IV. Manipulation of PPase levels for studies of PPase cellular function. With respect to I, our major goals are to: complete the determination of the relative orientation of the three or four divalent ions and two Pi's (or one PPi) bound per subunit of enzyme; identify additional amino acid residues at or near the active site; resolve the remaining ambiguities concerning the stereochemistry of enzyme catalysis; test the adequacy of our minimal scheme in accounting for overall PPase catalyses. With respect to II our goal is to isolate and sequence a PPase clone. In work on III, we will: use such a clone to express yeast PPase in a heterologous system; exploit such a system to produce site-specific mutants in PPase; examine the function of essential amino acid residues at the active site through functional studies on the mutant enzymes. Finally, in work on IV, we will exploit the PPase clone to construct yeast strains having both higher and lower levels of PPase compared to wild type. The phenotypes of such strains will then be tested in order to develop a convincing model of the cellular function of PPase.
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