On the Oxidative Half-Reaction of Plasmodium Falciparum Dihydroorotate Dehydogenase

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2007-12-18

Authors

Malmquist, Nicholas Anthony

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Plasmodium falciparum is the parasite responsible for an estimated 500 million malaria cases per year, which result in 1-2 million annual deaths. Current antimalarial chemotherapies are met with the tremendous ability of the parasite to develop resistance, underscoring the need for newer, more potent antimalarial drugs. Survival of the malaria parasite is dependent upon de novo biosynthesis of pyrimidines, as the organism is deficient in pyrimidine salvage. Dihydroorotate dehydrogenase (DHODH) is the flavoenzyme which catalyzes the fourth step in this pathway. The studies presented here describe the oxidative half-reaction of Pf DHODH where the enzyme, containing reduced falvin mononucleotide (FMN), is re-oxidized by terminal electron acceptors. The lipophilic co-substrate ubiquinone (CoQ) is shown to partition into detergent micelles in a hydrophobic chain length-dependent manner. Additionally, the enzyme is shown to associate with liposomes, which is likely mediated by its hydrophobic N-terminal domain. This arrangement reflects the physiological location of CoQ co-substrates within, and the attachment of the enzyme to, the inner mitochondrial membrane. Catalysis of CoQ analogues which partition into detergent micelles fit well to a surface dilution kinetic model, while catalysis of a CoQ analogue which remains in solution is well described by a solution steady-state kinetic model. These results suggest that the enzyme can perform catalysis of hydrophobic CoQ co-substrates at the surface-solution interface. Steady-state kinetic analysis of the complete Pf DHODH reaction cycle revealed only a modest alteration in the KM app for CoQ analogues upon mutation of several CoQ binding site residues to alanine, but displayed a more substantial effect on the catalytic rate upon mutation of a subset of residues. Pre-steady-state kinetic analysis showed both the dihydroorotate (DHO)-dependent half-reaction and the CoQ-dependent half-reaction to be faster than the observed steady-state rate. The A77 1726 binding-site mutations had no effect on the rate of the reductive half-reaction, but several reduced the rate of the CoQ-dependent flavin oxidation step without significantly altering the K,sub>d for CoQ. Inhibitors which bind in the proposed CoQ site block the CoQ-dependent oxidative half-reaction but do not inhibit the DHO-dependent reductive halfreaction. These results clearly distinguish the two co-substrate binding sites for DHO and CoQ and identify residues involved in electron transfer to physiologically relevant terminal electron acceptor substrates at a distant site.

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