In order to elucidate the molecular basis of diseases, there is a need to acquire a fundamental understanding of the relationship between protei structure and function at the molecular level. This knowledge will allow us to understand enzyme catalysis better, and make it possible to design specific inhibitors that can regulate enzyme activity. The model system to be used for this project is alkaline phosphatase, an enzyme that catalyzes the nonspecific hydrolysis of phosphate esters. Lack of activity of this enzyme results in the fatal hereditary disease hypophosphatasia, which is due to insufficient phosphate for bone calcification. In addition, alkaline phosphatase and the Ser/Thr phosphatases, which are involved in the metabolic control of a large number of important cellular processes, have a common intermediate in their mechanisms. We have selected the alkaline phosphatase from E. coli for this project because this system not only lends itself readily to time-resolved protein crystallographic studies, but also provides a unique system in which t investigate fundamental questions concerning the relationship between protein structure and function.
The specific aims of this revised proposal are to (i) use a combination of crystallographic techniques in combination with judicious choice of pH and temperature to determine the three-dimensional structures of the enzyme in the absence and presence of substrates at 1.75 angstroms, as well as the covalent and noncovalent enzyme-phosphate complexes, thus revealing subtle details abou each step in the reaction mechanism, (ii) determine the structures of the enzyme with a series of inhibitors bound so as to develop general rules for inhibition of the entire class of metallophosphatases, (iii) elucidate the importance of mobility of individual active site residues, (iv) continue to investigate the molecular basis of intragenic complementation, (v) determine the structure of mutants in which the active site serine has been replaced in order to learn more about phosphoester hydrolysis without a phosphoserine intermediate, and (vi) continue to elucidate the contribution of individual amino acids and the metals towards structural stabilization and catalysis. In addition, the crystallographic data will be used to (I) create a frame-by-fram movie showing how this prototypical phosphatase functions at the molecular level and (ii) develop leads for the design of second generation metallophosphatase inhibitors.
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