| Literature DB >> 20056101 |
Yasutoshi Kido1, Kimitoshi Sakamoto, Kosuke Nakamura, Michiyo Harada, Takashi Suzuki, Yoshisada Yabu, Hiroyuki Saimoto, Fumiyuki Yamakura, Daijiro Ohmori, Anthony Moore, Shigeharu Harada, Kiyoshi Kita.
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.Entities:
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Year: 2010 PMID： 20056101 DOI： 10.1016/j.bbabio.2009.12.021
Source DB: PubMed Journal: Biochim Biophys Acta ISSN： 0006-3002