It is the objective of this proposal to determine the high resolution X-ray crystal structure of inorganic pyrophosphatase from Bakers' yeast (PPase). This enzyme, which is essential in all cellular life, controls the level of pyrophosphate ion. This ion has a central role in the calcification of bones and teeth through its control of the rate of formation and growth of calcium phosphate crystals. Elevated levels of pyrophosphate ion have been found in the plasma and the synovial fluid of patients with osteoarthritis, pseudogout, acromegaly and uremia. PPase crytallizes in the space group P21 with one dimeric molecule of molecular weight 64,000 in the asymmetric unit of its unit cell. The diffraction pattern of the crystals exhibits significant presence out to at least a resolution corresponding to spacing of 2.5 angstrom. X-ray diffraction data of PPase crystals at a temperature of -40 degrees C are being recorded by rotation photography and measured with a scanning microdensitometer. The phases of the reflections will be determined using the technique of multiple isomorphous replacement. The high resolution three dimensional structure of the enzyme will be elucidated from consideration of the resulting electron density map together with the known amino acid sequence of the protein. The mode of substrate binding and any changes in protein conformation on binding the substrate will be determined from X-ray structural studies of enzyme-inhibitor complexes or a true enzyme-substrate complex stabilized at low temperatures. This structural information, when taken together with chemical and kinetic data, should indicate the catalytic mechanism of PPase. This, in turn, should lead to a greater understanding of normal and pathological states of mineralized tissues and hopefully to the development of specific chemotherapeutic agents for diseases of such tissues.