Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei
Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei
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DOI：
10.1016/j.bbabio.2009.12.021
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发表时间：
2010-04-01
影响因子：
4.3
通讯作者：
Kita, Kiyoshi
中科院分区：
文献类型：
--
作者：
Kido, Yasutoshi;Sakamoto, Kimitoshi;Kita, Kiyoshi
锥虫交替氧化酶（TAO）在非洲锥虫中作为细胞色素非依赖性末端氧化酶发挥功能，其对于它们在哺乳动物宿主中的存活是必需的，并且由于其不存在于哺乳动物宿主中，因此被认为是治疗锥虫病的有前景的药物靶标。在本研究中，重组TAO（rTAO）过表达的血缺陷大肠杆菌菌株已被溶解的E。大肠杆菌膜，并纯化至均匀的稳定和高活性的形式。通过电感耦合等离子体质谱仪（ICP-MS）检测的结合铁的分析揭示了每个rTAO单体两个结合铁原子的化学计量。通过EPR分析证实rTAO确实是二铁蛋白，EPR分析显示rTAO还原形式的信号，g值为15。纯化的rTAO氧化泛醇-1的动力学显示典型的Michaelis-Menten动力学（Km为338 μ M，V-max为601 μ mol/min/mg），而泛醇-2氧化显示不寻常的底物抑制。特异性抑制剂，ascofuranone，抑制酶的混合型抑制的方式相对于泛醇-1。(C)2009 Elsevier B. V.保留所有权利。
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 338 mu M and V-max of 601 mu mol/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. (C) 2009 Elsevier B.V. All rights reserved.