Wiseman J S
Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Biochemistry. 1989 Mar 7;28(5):2106-11. doi: 10.1021/bi00431a021.
Isotope effects for the oxidation of [5,6,8,9,11,12,14,15-3H]arachidonic acid catalyzed by soybean lipoxygenase and by 5-lipoxygenase were measured. This labeling pattern represents substitution at each of the vinylic hydrogens of the substrate. The observed isotope effect for soybean lipoxygenase was 1.16 +/- 0.02 and for 5-lipoxygenase was 1.11 +/- 0.05. These isotope effects are inconsistent with any change in hybridization (sp2 to sp3) at the vinylic carbons prior to or during the rate-determining step and are concluded to be most consistent with the formation of a carbanion-like intermediate or transition state. In contrast, the oxidation of arachidonic acid by Ce(IV), which is thought to proceed via a cation radical intermediate, exhibited at most a small isotope effect (1.02 +/- 0.01). The reduction potential for the cation radical formed from arachidonic acid in this reaction is estimated to be 2.7 V vs NHE by comparison of the rates of oxidation of arachidonic acid and cyclohexene by Ce(IV). This is similar to the potential for the cation radical of 2-butene. No isotope effect (1.00 +/- 0.03) was observed in the 5-lipoxygenase reaction for conversion of the initially formed product 5-hydroperoxyeicosatetraenoic acid to the epoxide leukotriene A4. From this it is concluded that there is little carbon-oxygen bond formation prior to or during the rate-determining step for epoxide formation.
测定了大豆脂氧合酶和5-脂氧合酶催化的[5,6,8,9,11,12,14,15-³H]花生四烯酸氧化的同位素效应。这种标记模式代表底物每个烯丙基氢的取代。观察到大豆脂氧合酶的同位素效应为1.16±0.02，5-脂氧合酶的同位素效应为1.11±0.05。这些同位素效应与限速步骤之前或期间烯丙基碳上的杂化变化（sp²到sp³）不一致，得出的结论是，它们与类碳负离子中间体或过渡态的形成最一致。相比之下，铈（IV）催化的花生四烯酸氧化，被认为是通过阳离子自由基中间体进行的，其同位素效应至多很小（1.02±0.01）。通过比较铈（IV）对花生四烯酸和环己烯的氧化速率，估计该反应中花生四烯酸形成的阳离子自由基的还原电位相对于标准氢电极（NHE）为2.7V。这与2-丁烯阳离子自由基的电位相似。在5-脂氧合酶反应中，最初形成的产物5-氢过氧化二十碳四烯酸转化为环氧化物白三烯A4时未观察到同位素效应（1.00±0.03）。由此得出结论，在环氧化物形成的限速步骤之前或期间，几乎没有碳-氧键形成。
