PYRUVATE PHOSPHATE DIKINASE

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DESCRIPTION (Adapted from the applicant's abstract): This application request funds to continue investigations into the structure and catalytic mechanism of the enzyme pyruvate phosphate dikinase (PPDK). The knowledge gained from these studies will (1) serve as the foundation for the design of drugs to combat human infection by parasites and (2) will provide special insight into the mechanisms and mechanics/dynamics of ligand modulated protein motion. During the next funding period investigators intend to focus on the active site regions of PPDK and specifically, on the structural and electronic changes that occur at these sites during catalytic turnover. This knowledge will expand understanding of catalysis in phosphoryl transfer reactions and, ultimately will serve as the foundation for the design of tight binding inhibitors specific for the PPDK active sites. The specific aims for this phase of the project are to: determine the active site residues which function in metal ion cofactor and substrate binding, in acid/base catalysis and in transition state/intermediate binding and to determine the role of the metal ion cofactors in substrate binding and catalysis. During the next funding period investigators will also examine how domain movement in PPDK provides for the transport of the catalytic histidine between the separate active sites in the enzyme during catalytic turnover. This knowledge will expand our understanding of how catalysis at separate active sites in multienzyme complexes is mediated by an appended carrier ligand (lipoate, pantethiene). The specific aims for this phase of the project are to: define the enzyme conformation assumed during each of the three partial reactions, to deduce the torsional angles altered during the interconversion of the conformers, to pin-point the noncovalent bonding interactions which serve to stabilize the respective conformational states and to measure the rate at which the conformers interconvert and the dependence of the rate on the structural features of the protein.