Mechanisms of Dihydroorotate Dehydrogenases

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DESCRIPTION: (provided by applicant) Reaction mechanisms will be determined for the family 1A dihydroorotate dehydrogenase (DHOD) from Lactococcus lactis and Enterococcus faecalis, and the family 2 enzymes from Homo sapiens and Escherichia coli. DHOD, the only redox enzyme in pyrimidine biosynthesis, is an attractive target for drug design in the treatment of many diseases, including malaria, arthritis, and Pneumocystis infections in AIDS patients. The mechanism of reduction of the enzyme-bound FMN by dihydroorotate for the two enzymes will be determined under anaerobic conditions through stopped-flow kinetic analyses, including the use of single- and double deuterium substrate isotope effects, solvent isotope effects, and the pH dependence of the rate constants. Mutant L. lactis A and E. coli enzymes will be studied in order to determine the roles that active site residues have in the reaction. The mechanism of oxidation of reduced L. lactis DHOD A by fumarate will be elucidated in stopped-flow experiments that will determine the pH dependence of the reaction, and in double-mixing experiments, the deuterium isotope effects for the transfer of each hydrogen to fumarate. The mechanism(s) of oxidation by quinones of DHOD A and the E. coli enzyme will be determined in stopped-flow experiments utilizing a range of quinone substrates. The ubiquinone isoprenyl chain-length preference of the E. coli enzyme will be determined. Enzyme-ligand interactions will be probed with Raman spectroscopy.