INVESTIGATION OF PYRUVATE PHOSPHATE DIKINASE

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The overall goal of this project is to construct a detailed picture of (i) the active site regions where the three partial reactions comprising the PPDK reaction take place, (ii) the mechanisms by which these three partial reactions take place and (iii) the mechanism by which the putative histidine phosphoryl carrier shuttles from one active site region to the next. The specific aims of the project are: (1) To sequence the Bacteriodes symbiosus PPDK gene. (2) To crystallize PPDK and initiate X-ray crystallographic studies. (3) To determine the rate constants for the PPDK substrate binding and release steps and catalytic steps and to construct a free energy profile for the PPDK reaction. (4) To determine the stoichiometry, location and role of the metal ion cofactors during PPDK catalysis. (5) To identify the PPDK active site amino acid residues involved in substrate binding and catalysis. (6) To examine the structure of the active with regard to overlapping vs nonoverlapping sites for the three partial reactions and to test for movement of the putative "phosphoryl carrier histidine" between reaction sites during catalysis.