Purification and kinetic characterisation of recombinant alternative oxidase from Trypanosoma brucei brucei

Kido, Yasutoshi, Sakamoto, Kimitoshi, Nakamura, Kosuke, Harada, Michiyo, Suzuki, Takashi, Yabu, Yoshisada, Saimoto, Hiroyuki, Yamakura, Fumiyuki, Ohmori, Daijiro, Moore, Anthony, Harada, Shigeharu and Kita, Kiyoshi (2010) Purification and kinetic characterisation of recombinant alternative oxidase from Trypanosoma brucei brucei. BBA - Bioenergetics, 1797 (4). pp. 443-450. ISSN 0005-2728

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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 (Km of 338 µM and Vmax of 601 µmol/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.

Item Type: Article
Schools and Departments: School of Life Sciences > Biochemistry
Depositing User: EPrints Services
Date Deposited: 06 Feb 2012 20:02
Last Modified: 11 Jun 2012 09:26
URI: http://sro.sussex.ac.uk/id/eprint/23723
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