<br>脂质组学揭示多不饱和脂肪酸离体酶促氧化过程中显着的生理动力学同位素效应,Journal of the American Chemical Society

当前位置： X-MOL 学术 › J. Am. Chem. Soc. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

脂质组学揭示多不饱和脂肪酸离体酶促氧化过程中显着的生理动力学同位素效应
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2017-12-19 , DOI: 10.1021/jacs.7b09493
Aaron R Navratil ₁ , Mikhail S Shchepinov ₂ , Edward A Dennis ₁

Affiliation

花生四烯酸（AA，20:4）是一种 omega-6 多不饱和脂肪酸 (PUFA)，也是类二十烷酸类脂质介质的主要前体。催化 AA 氧化的酶首先从 1,4-顺式,顺式二烯单元内的三个双烯丙基碳之一中提取氢。已证明用氘替代氢可对亚油酸的大豆脂氧合酶 (sLOX) 氧化产生巨大的动力学同位素效应 (KIE)（LA，18:2）。然而，在哺乳动物酶（包括环氧合酶（COX）和脂氧合酶（LOX））对 AA 和 LA 进行氧化过程中 KIE 的实验测定结果显示，其值要低得多。所有先前研究 PUFA 氧化 KIE 的研究都依赖于使用纯化酶的体外系统，并且受到氘代底物可用性的限制。在这里，我们展示了使用巨噬细胞作为离体模型系统，使用新合成的氘代 AA 同位素体库来研究活细胞酶促 AA 氧化过程中的生理 KIE (PKIE)。通过扩展脂质组学 UPLC-MS/MS 方法来同时定量天然和氘代脂质产物，我们能够证明在巨噬细胞中测量的 AA 的 COX 和 LOX 氧合的 PKIE 的大小与之前报告中使用 AA 确定的 KIE 相似同位素异构体在碳 13 (C13) 处氘化。然而，我们首次表明，增加氘代双烯丙基碳的数量以包括 C10 和 C13 会导致 AA 的 COX 氧化的 PKIE 大幅增加。我们提供的证据表明，AA C10 上存在的氢在前列腺素和血栓素合成的催化中发挥着关键作用。此外，我们发现 C10 的氘化可能通过在生理条件下干扰 AA 环化并将 AA 分流至 LOX 途径来促进溶解脂质介质脂氧素 B4 的形成。

"点击查看英文标题和摘要"

Lipidomics reveals dramatic physiological kinetic isotope effects during the enzymatic oxygenation of polyunsaturated fatty acids ex vivo

Arachidonic acid (AA, 20:4) is an omega-6 polyunsaturated fatty acid (PUFA) and the main precursor to the class of lipid mediators known as eicosanoids. The enzymes that catalyze the oxygenation of AA begin by abstracting hydrogen from one of three bis-allylic carbons within 1,4-cis,cis-diene units. Substitution of deuterium for hydrogen has been shown to lead to massive kinetic isotope effects (KIE) for soybean lipoxygenase (sLOX) oxygenation of linoleic acid (LA, 18:2). Yet, experimental determination of the KIE during oxygenation of AA and LA by mammalian enzymes including cyclooxygenase (COX) and lipoxygenase (LOX) has revealed far lower values. All prior studies investigating the KIE of PUFA oxygenation have relied on in vitro systems using purified enzymes and were limited by availability of deuterated substrates. Here we demonstrate the use of macrophages as an ex vivo model system to study the physiological KIE (PKIE) during enzymatic AA oxygenation by living cells using a newly synthesized library of deuterated AA isotopologues. By extending lipidomic UPLC-MS/MS approaches to simultaneously quantify native and deuterated lipid products, we were able to demonstrate that the magnitude of the PKIE measured in macrophages for COX and LOX oxygenation of AA is similar to KIEs determined in previous reports using the AA isotopologue deuterated at carbon 13 (C13). However, for the first time we show that increasing the number of deuterated bis-allylic carbons to include both C10 and C13 leads to a massive increase in the PKIE for COX oxygenation of AA. We provide evidence that hydrogen(s) present at C10 of AA play a critical role in the catalysis of prostaglandin and thromboxane synthesis. Furthermore, we discovered that deuteration of C10 promotes the formation of the resolving lipid mediator lipoxin B4, likely by interfering with AA cyclization and shunting AA to the LOX pathway under physiological conditions.

更新日期：2017-12-19

点击分享查看原文

点击收藏

公开下载

阅读更多本刊新发论文本刊介绍/投稿指南