Probing the ubiquinol oxidatiousite of Eschenchia col ubiquinol oxidases.

底物结合位点的大肠杆菌bo-和BD-型醌醇氧化酶探测与系统合成的泛醇类似物。泛醇-2衍生物对bo型酶的表观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，不仅2-位的甲氧基比3-位的甲氧基更严格地被结合位点识别，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。在与bd型酶的平行研究中，我们获得了类似的观察结果，除了几乎所有的醌醇类似物，但不是泛醇-1，在较高浓度下引起显着的底物抑制。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。

Substrate binding sites of the Escherichia coli bo-and bd-type quinol oxidases were probed with systematically synthesized ubiquinol analogues. The apparent K_m values of ubiquinol-2derivatives to the bo-type enzyme 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 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 bd-type enzyme, we obtained similar observations except that almost all quinol analogues, but not ubiquinol-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.