Isotope sensitive branching and kinetic isotope effects in the reaction of deuterated arachidonic acids with human 12- and 15-lipoxygenases
Isotope sensitive branching and kinetic isotope effects in the reaction of deuterated arachidonic acids with human 12- and 15-lipoxygenases
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DOI：
10.1021/bi800308q
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发表时间：
2008-07-08
期刊：
影响因子：
2.9
通讯作者：
van der Donk, Wilfred A.
中科院分区：
文献类型：
--
作者：
Jacquot, Cyril;Wecksler, Aaron T.;van der Donk, Wilfred A.
脂氧合酶 (LO) 催化脂质过氧化，并与许多与氧化应激和炎症有关的人类疾病有关。由于以亚油酸 (LA) 作为底物的限速氢提取步骤的大动力学同位素效应 (30-80)，这些酶也引起了相当大的关注。在此，我们报告了三种人类 LO（血小板 12-hLO、网织红细胞 15-hLO-1 和上皮细胞 15-hLO-2）与花生四烯酸 (AA) 反应中的动力学同位素效应 (KIE)。令人惊讶的是，观察到的 AA 的 KIE 比之前报道的 LA 的值小得多。对较小 KIE 起源的研究导致了反应途径同位素敏感分支的发现。产物分布分析表明 15-hLO-1 的区域选择性发生反转，未标记 AA 中从 C13 夺氢是主要途径，但当 13 位亚甲基被氘化时，从 C10 夺氢占主导地位。 12-hLO 和 15-hLO-2 的区域选择性也发生了较小但明显的变化。
Lipoxygenases (LOs) catalyze lipid peroxidation and have been implicated in a number of human diseases connected to oxidative stress and inflammation. These enzymes have also attracted considerable attention due to large kinetic isotope effects (30-80) for the rate-limiting hydrogen abstraction step with linoleic acid (LA) as substrate. Herein, we report kinetic isotope effects (KIEs) in the reactions of three human LOs (platelet 12-hLO, reticulocyte 15-hLO-1, and epithelial 15-hLO-2) with arachidonic acid (AA). Surprisingly, the observed KIEs with AA were much smaller than the previously reported values with LA. Investigation into the origins for the smaller KIEs led to the discovery of isotope sensitive branching of the reaction pathways. Product distribution analysis demonstrated an inversion in the regioselectivity of 15-hLO-1, with hydrogen abstraction from C13 being the major pathway with unlabeled AA but abstraction from C10 predominating when the methylene group at position 13 was deuterated. Smaller but clear changes in regioselectivity were also observed for 12-hLO and 15-hLO-2.