Our major goal is to use site-specific mutagenesis together with rate, equilibrium, and structural studies to understand the basis for PPase catalysis of PPi hydrolysis by fully characterizing the active site structure and catalytic mechanism of S. cerevisiae PPase (Y1-PPase) and E. coli PPase (E-PPase). Phosphoryl transfer enzymes form one of the largest classes of enzymes in Nature, yet their mechanisms of action remain incompletely understood. Success in achieving our goal will provide a detailed understanding of the mechanism of action of an important member of this class and allow us to test the proposition that the catalytic mechanism and active site of soluble PPases are largely conserved evolutionarily. This work will involve the study of Y1-PPase and E-PPase variants mutated at putative site residues including: a) kinetic, binding, and stereochemical investigations directed toward determination of kinetic schemes, microscopic rate constants, pH-rate profiles, ligand [M2+, inorganic phosphate (Pi)] binding constants and stoichiometry; b) structural studies, including circular dichroism, dye binding, and thermal stability directed toward determining whether effects seen on mutation are limited to changes in the active site or rather reflect larger structural changes in the enzyme; c) NMR and EPR experiments directed toward measuring M2+:phosphoryl ligand and M2+:M2+ interactions at the enzyme active site; d) the preparation of crystals of E-PPase and Y1-PPase variants suitable for X-ray crystallographic analysis. The results of such studies, interpreted in the light of ongoing studies of the three-dimensional structures of both enzymes, will permit us to determine how site-specific mutations affect PPase. They also will reveal whether mutations at residues that are identical in Y1-PPase and E-PPase have the same relative effects on function and/or structure, thus permitting an important test of the notion of a conserved active site and mechanism for PPases. Successful completion of the work herein proposed will have three important consequences. First, it will provide a model for the mechanisms of other phosphoryl transfer enzymes that use several metal ions at the active site. Second, by exploring the differences in detail between the mechanism of procaryotic PPases, represented by E-PPase, and of eucaryotic PPases, represented by Y1-PPase, it may allow for the development of therapeutic agents that exploit these differences. Third, it could have important consequences for the treatment of calcium pyrophosphate dihydrate (CPPD) crystal deposition disease. A disorder of PPi metabolism is suspected in the above-mentioned disease. Recent work on bovine retinal PPase makes it likely that the human PPA clone will soon be available. The availability of this clone would provide an important tool for the further study and/or treatment of CPPD crystal deposition disease e.g., by gene therapy. Our detailed studies on Y1-PPase might suggest human PPase variants of heightened therapeutic effectiveness (e.g., by manipulation of pH optima, of Km values).

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Biochemistry Study Section (BIO)
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University of Pennsylvania
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