Hoffmann Inga, Hamberg Mats, Lindh Roland, Oliw Ernst H
Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
Biochim Biophys Acta. 2012 Dec;1821(12):1508-17. doi: 10.1016/j.bbalip.2012.09.001. Epub 2012 Sep 12.
Cyclooxygenases (COX) and 8R-dioxygenase (8R-DOX) activities of linoleate diol synthases (LDS) are homologous heme-dependent enzymes that oxygenate fatty acids by a tyrosyl radical-mediated hydrogen abstraction and antarafacial insertion of O(2). Soybean lipoxygenase-1 (sLOX-1) contains non-heme iron and oxidizes 18:2n-6 with a large deuterium kinetic isotope effect (D-KIE). The aim of the present work was to obtain further mechanistic insight into the action of these enzymes by using a series of n-6 and n-9 fatty acids and by analysis of D-KIE. COX-1 oxidized C(20) and C(18) fatty acids in the following order of rates: 20:2n-6>20:1n-6>20:3n-9>20:1n-9 and 18:3n-3≥18:2n-6>18:1n-6. 18:2n-6 and its geometrical isomer (9E,12Z)18:2 were both mainly oxygenated at C-9 by COX-1, but the 9Z,12E isomer was mostly oxygenated at C-13. A cis-configured double bond in the n-6 position therefore seems important for substrate positioning. 8R-DOX oxidized (9Z,12E)18:2 at C-8 in analogy with 18:2n-6, but the 9E,12Z isomer was mainly subject to hydrogen abstraction at C-11 and oxygen insertion at C-9 by 8R-DOX of 5,8-LDS. sLOX-1 and 13R-MnLOX oxidized [11S-(2)H]18:2n-6 with similar D-KIE (~53), which implies that the catalytic metals did not alter the D-KIE. Oxygenation of 18:2n-6 by COX-1 and COX-2 took place with a D-KIE of 3-5 as probed by incubations of [11,11-(2)H(2)]- and [11S-(2)H]18:2n-6. In contrast, the more energetically demanding hydrogen abstractions of the allylic carbons of 20:1n-6 by COX-1 and 18:1n-9 by 8R-DOX were both accompanied by large D-KIE (>20).
亚油酸二醇合酶（LDS）的环氧化酶（COX）和8R-双加氧酶（8R-DOX）活性是同源的血红素依赖性酶，它们通过酪氨酸自由基介导的氢提取和O₂的反式插入来氧化脂肪酸。大豆脂氧合酶-1（sLOX-1）含有非血红素铁，并以较大的氘动力学同位素效应（D-KIE）氧化18:2n-6。本研究的目的是通过使用一系列n-6和n-9脂肪酸并分析D-KIE，进一步深入了解这些酶的作用机制。COX-1氧化C₂₀和C₁₈脂肪酸的速率顺序如下：20:2n-6>20:1n-6>20:3n-9>20:1n-9和18:3n-3≥18:2n-6>18:1n-6。18:2n-6及其几何异构体（9E,12Z）18:2主要在C-9位被COX-1氧化，但9Z,12E异构体大多在C-13位被氧化。因此，n-6位的顺式双键似乎对底物定位很重要。8R-DOX与18:2n-6类似，在C-8位氧化（9Z,12E）18:2，但9E,12Z异构体主要在C-11位发生氢提取，并被5,8-LDS的8R-DOX在C-9位插入氧。sLOX-1和13R-MnLOX以相似的D-KIE（约53）氧化[11S-(2)H]18:2n-6，这意味着催化金属没有改变D-KIE。通过[11,11-(2)H₂]-和[11S-(2)H]18:2n-6的孵育检测，COX-1和COX-2氧化18:2n-时的D-KIE为3-5。相比之下，COX-1对20:1n-6的烯丙基碳和8R-DOX对18:1n-9的烯丙基碳进行的能量需求更高的氢提取都伴随着较大的D-KIE（> 20）。
