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Study on ubiquinol reaction site of cytochrome bo in Escherichia coli.

大肠杆菌细胞色素bo泛醇反应位点的研究

基本信息

批准号：
11660108
负责人：
MIYOSHI Hideto
金额：
$ 2.11万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660108/
关键词：
E.coli terminal oxidese Respiratory enzymes Ubiquinone Structure-activity relation Aurachin Respiratory inhibitors

项目摘要

Substrate binding sites of the Escherichia coli cytochromes bo and bd were probed with systematically synthesized ubiquinol analogues. The apparent K_m values of Q_2H_2 derivatives to the cytochrome bo were much lower than that of the corresponding 6-n-decyl derivatives. The isoprenoid structure is less hydrophobic than the saturated n-alkyl group with the same carbon number, therefore, the native isoprenoid side chain appears to play a specific role in quinol binding besides simply increasing hydrophobicity of the molecule. The V_<max> values of 2-methoxy-3-ethoxy analogues were greater than that of 2-ethoxy-3-methoxy analogues irrespective of the side chain structure. This result indicates not only that a methoxy group in the 2-position is recognized more strictly than the 3-position by the binding site, but also that the side chain structure does not affect binding of the quinol ring moiety. Systematic analysis of the electron-donating activities of the analogues with different substituents in the 5-position revealed that the 5-methyl group is important for the activity. In the parallel studies with the cytochrome bd, similar observations were obtained except that almost all quinol analogues, but not Q_1, elicited a remarkable substrate inhibition at higher concentrations. These results indicate that the structurally unrelated two terminal oxidases share common structural properties for the quinol oxidation site. In addition, to investigate the quinone binding site of cytochrome bo enzyme, convenient synthetic procedures which enable the preparation of photolable 2-azido-Q_2 and 3-azido-Q_2 have been established.

底物结合位点的大肠杆菌细胞色素bo和bd探测与系统合成的泛醇类似物。Q_2H_2衍生物对细胞色素bo的表观K_m值远低于相应的6-正癸基衍生物。类异戊二烯结构的疏水性低于具有相同碳数的饱和正烷基，因此，天然类异戊二烯侧链似乎除了简单地增加分子的疏水性之外，还在醌醇结合中发挥特定作用。无论<max>侧链结构如何，2-甲氧基-3-乙氧基类似物的V_值均大于2-乙氧基-3-甲氧基类似物。该结果表明，结合位点不仅比3位更严格地识别2位的甲氧基，而且侧链结构不影响喹啉环部分的结合。对5-位上不同取代基的类似物的供电子活性进行了系统分析，发现5-甲基对供电子活性有重要影响。在细胞色素bd的平行研究中，除了几乎所有的醌醇类似物在较高浓度下引起显著的底物抑制外，获得了类似的观察结果，但Q_1没有。这些结果表明，结构上不相关的两个终端氧化酶的醌醇氧化位点有共同的结构特性。此外，为了研究细胞色素bo酶的醌结合位点，建立了简便的合成方法，使光稳定的2-叠氮基-Q_2和3-叠氮基-Q_2的制备成为可能。