Analysis of Flavin Oxidation and Electron-Transfer Inhibition in Plasmodium falcipa rum Dihydroorotate Dehydrogenase.

Plasmodium jalciparum dihydroorotate dehydrogenase (pJDHOL)H) is a tiavin-dependent mitochondrial enzyme that provides the only route to pyrimidine biosynthesis in the parasite. Clinically significant inhibitors of human DHODH (e.g., A77 1726) bind to a pocket on the opposite face of the flavin cofactor from dihydroorotate (DHO). This pocket demonstrates considerable sequence variability, which has allowed species-specific inhibitors of the malarial enzyme to be identified. Ubiquinone (CoQ), the physiological oxidant in the reaction, has been postulated to bind this site despite a lack of structural evidence. To more clearly define the residues involved in C0Q binding and catalysis, we undertook site- directed mutagenesis of seven residues in the structurally defined A77 1726 binding site, which we term the species-selective inhibitor site. Mutation of several of these residues (HI 85, Fl 88, and F227) to Ala substantially decreased the affinity of pJDHODH-specific inhibitors (40-240-fold). In contrast, only a modest increase in the KmaPP for C0Q was observed, although mutation of Y528 in particular caused a substantial reduction in kcat (40-100-fold decrease). Pre-steady-state kinetic analysis by single wavelength stopped-flow spectroscopy showed that the mutations had no effect on the rate of the DHO-dependent reductive half-reaction, but most reduced the rate of the CoQ-dependent flavin oxidation step (3-20-fold decrease), while not significantly altering the Kd0'~ for C0Q. As with the mutants, inhibitors that bind this site block the C0Q-dependent oxidative half-reaction without affecting the DHO-dependent step. These results identify residues involved in inhibitor binding and electron transfer to CoQ. Importantly, the data provide compelling evidence that the binding sites for C0Q and species-selective site inhibitors do not overlap, and they suggest instead that inhibitors act either by blocking the electron path between flavin and C0Q or by stabilizing a conformation that excludes CoQ binding. [ABSTRACT FROM AUTHOR]