Probing the ubiquinol oxidatiousite of Eschenchia col ubiquinol oxidases.

用系统合成的泛醇类似物探测了大肠杆菌b型和b型喹啉氧化酶的底物结合位点。泛醇-2衍生物对o型酶的表观K_m值远低于相应的6-n-癸基衍生物。与相同碳数的饱和正烷基相比，类异戊二烯结构的疏水性较差，因此，除了简单地增加分子的疏水性外，天然的类异戊二烯侧链似乎在喹啉结合中起着特殊的作用。无论侧链结构如何，2-甲氧基-3-乙氧基类似物的V_<max>值均大于2-乙氧基-3-甲氧基类似物。这一结果不仅表明2位的甲氧基比3位的结合位点更严格地识别，而且表明侧链结构不影响喹啉环部分的结合。系统分析了具有不同5位取代基的类似物的给电子活性，发现5-甲基对活性起重要作用。在与bd型酶的平行研究中，我们得到了类似的观察结果，除了几乎所有的喹诺类似物，但泛醇-1不存在，在较高浓度下引起显着的底物抑制。这些结果表明，结构无关的两个末端氧化酶对喹啉氧化位点具有共同的结构性质。

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.