title: Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei.
abstract: The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K(m) of 338microM and V(max) of 601micromol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.
authors.affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Yasutoshi
authors.initials: Y
authors.lastname: Kido
authors.forename: Kimitoshi
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Kosuke
authors.initials: K
authors.lastname: Nakamura
authors.forename: Michiyo
authors.initials: M
authors.lastname: Harada
authors.forename: Takashi
authors.initials: T
authors.lastname: Suzuki
authors.forename: Yoshisada
authors.initials: Y
authors.lastname: Yabu
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Saimoto
authors.forename: Fumiyuki
authors.initials: F
authors.lastname: Yamakura
authors.forename: Daijiro
authors.initials: D
authors.lastname: Ohmori
authors.forename: Anthony
authors.initials: A
authors.lastname: Moore
authors.forename: Shigeharu
authors.initials: S
authors.lastname: Harada
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Kita
mesh_terms: D002384:Catalysis / Q000187:drug effects; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D013058:Mass Spectrometry; D024101:Mitochondrial Proteins; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012717:Sesquiterpenes / Q000494:pharmacology; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism; D000099188:Alternative Oxidase
chemical_list: D004791:Enzyme Inhibitors; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D014451:Ubiquinone; D000099188:Alternative Oxidase; C006640:ascofuranone; C003741:ubiquinol