title: Detergent-dependent kinetics of truncated Plasmodium falciparum dihydroorotate dehydrogenase.
abstract: The survival of the malaria parasite Plasmodium falciparum is dependent upon the de novo biosynthesis of pyrimidines. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the fourth step in this pathway in an FMN-dependent reaction. The full-length enzyme is associated with the inner mitochondrial membrane, where ubiquinone (CoQ) serves as the terminal electron acceptor. The lipophilic nature of the co-substrate suggests that electron transfer to CoQ occurs at the two-dimensional lipid-solution interface. Here we show that PfDHODH associates with liposomes even in the absence of the N-terminal transmembrane-spanning domain. The association of a series of ubiquinone substrates with detergent micelles was studied by isothermal titration calorimetry, and the data reveal that CoQ analogs with long decyl (CoQ(D)) or geranyl (CoQ(2)) tails partition into detergent micelles, whereas that with a short prenyl tail (CoQ(1)) remains in solution. PfDHODH-catalyzed reduction of CoQ(D) and CoQ(2), but not CoQ(1), is stimulated as detergent concentrations (Tween 80 or Triton X-100) are increased up to their critical micelle concentrations, beyond which activity declines. Steady-state kinetic data acquired for the reaction with CoQ(D) and CoQ(2) in substrate-detergent mixed micelles fit well to a surface dilution kinetic model. In contrast, the data for CoQ(1) as a substrate were well described by solution steady-state kinetics. Our results suggest that the partitioning of lipophilic ubiquinone analogues into detergent micelles needs to be an important consideration in the kinetic analysis of enzymes that utilize these substrates.
authors.affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Malmquist
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Baldwin
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Phillips
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000090722:Dihydroorotate Dehydrogenase; D004579:Electron Transport; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D008823:Micelles; D008956:Models, Chemical; D017830:Octoxynol / Q000737:chemistry*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000235:genetics; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011136:Polysorbates / Q000737:chemistry*; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics; D017384:Sequence Deletion; D014451:Ubiquinone / Q000737:chemistry
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D008081:Liposomes; D008823:Micelles; D011136:Polysorbates; D015800:Protozoan Proteins; D014451:Ubiquinone; D017830:Octoxynol; D044925:Oxidoreductases Acting on CH-CH Group Donors; C025203:Ubiquinone Q1