Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei.
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.
Kido
Yasutoshi
Y
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Sakamoto
Kimitoshi
K
Nakamura
Kosuke
K
Harada
Michiyo
M
Suzuki
Takashi
T
Yabu
Yoshisada
Y
Saimoto
Hiroyuki
H
Yamakura
Fumiyuki
F
Ohmori
Daijiro
D
Moore
Anthony
A
Harada
Shigeharu
S
Kita
Kiyoshi
K
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
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