Ivanov I, Rathmann J, Myagkova G, Kuhn H
Institute of Biochemistry, University Clinics (Charité), Humboldt University, Hessische Strasse 3-4, 10115 Berlin, Germany.
Biochemistry. 2001 Aug 28;40(34):10223-9. doi: 10.1021/bi010700w.
The positional specificity is the decisive enzyme property for classification of lipoxygenases and for the currently used lipoxygenase nomenclature. It has been reported before that soybean lipoxygenase-1, which oxygenates polyenoic fatty acids at alkaline pH to the corresponding n - 6 hydroperoxy derivative, exhibits a different positional specificity when either the reaction conditions or the substrate structure is altered. To investigate the impact of structural substrate modifications on the positional specificity of this enzyme and to force an inverse substrate binding, we synthesized arachidonic acid analogues modified at the omega-terminus. Care was taken that the double bond system remained unchanged so that hydrogen abstraction from all three bisallylic methylenes was theoretically possible. We found that omega-modification of arachidonic acid leads to an impaired substrate affinity and a reduced reaction rate, but we did not detect any 5-lipoxygenation products, suggesting that structural modification of the omega-end may not be sufficient to force an inverse substrate orientation. However, when both ends of the fatty acid chain (omega-terminus and free carboxylate) were modified simultaneously, a considerable share of 5-lipoxygenation products was detected. These results indicate that introduction of polar or bulky groups at the methyl terminus of polyenoic fatty acids was not sufficient to force an inverse substrate orientation. However, simultaneous introduction of an omega-OH group and methylation of the carboxylate led to formation of significant 5-lipoxygenation products, suggesting an inverse head to tail substrate orientation.
位置特异性是脂氧合酶分类及当前使用的脂氧合酶命名法的决定性酶特性。之前有报道称，大豆脂氧合酶-1在碱性pH条件下将多烯脂肪酸氧化为相应的n - 6氢过氧化物衍生物，当反应条件或底物结构改变时，其表现出不同的位置特异性。为了研究底物结构修饰对该酶位置特异性的影响并促使底物反向结合，我们合成了在ω-末端修饰的花生四烯酸类似物。注意双键系统保持不变，以便理论上从所有三个双烯丙基亚甲基上夺取氢成为可能。我们发现花生四烯酸的ω-修饰导致底物亲和力受损和反应速率降低，但未检测到任何5-脂氧合产物，这表明ω-末端的结构修饰可能不足以促使底物反向定向。然而，当脂肪酸链的两端（ω-末端和游离羧酸盐）同时修饰时，检测到相当比例的5-脂氧合产物。这些结果表明，在多烯脂肪酸的甲基末端引入极性或庞大基团不足以促使底物反向定向。然而，同时引入ω-OH基团和羧酸盐甲基化导致形成大量的5-脂氧合产物，表明底物存在反向的头对尾定向。
