Novel insights into cyclooxygenases, linoleate diol synthases, and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs
Novel insights into cyclooxygenases, linoleate diol synthases, and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs
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DOI：
10.1016/j.bbalip.2012.09.001
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发表时间：
2012-12-01
影响因子：
4.8
通讯作者：
Oliw, Ernst H.
中科院分区：
文献类型：
--
作者：
Hoffmann, Inga;Hamberg, Mats;Oliw, Ernst H.
亚油酸二醇脱氢酶（LDS）的环加氧酶（考克斯）和8 R-双加氧酶（8 R-DOX）是一种同源的血红素依赖性酶，它们通过血红素氧化脂肪酸。酪氨酰自由基介导的氢提取和O-2的对面插入。大豆脂氧合酶-1（sLOX-1）含有非血红素铁，能氧化18：2n - 6，具有很强的氘动力学同位素效应（D-KIE）。本工作的目的是通过使用一系列n - 6和n - 9脂肪酸和通过D-KIE分析来获得对这些酶的作用的进一步的机理洞察。考克斯-1氧化C-20和C-18脂肪酸的速率顺序为：20：2n - 6>20：1 n- 6>20：3 n- 9>20：1 n- 9和18：3 n- 3 >= 18：2n - 6> 18：1 n- 6。18：2 n- 6及其几何异构体（9 E，12 Z）18：2均主要在C-9被考克斯-1氧化，而9 Z，12 E异构体主要在C-13被氧化。因此，在n - 6位的顺式构型双键对于底物定位似乎很重要。与18：2n - 6类似，8 R-DOX在C-8处氧化（9 Z，12 E）18：2，但9 E，12 Z异构体主要在C-11处被夺氢，并在C-9处被5，8-LDS的8 R-DOX插入氧。sLOX-1和13 R-MnLOX以类似的D-KIE（类似于53）氧化[11 S-H-2]18：2n - 6，这意味着催化金属没有改变D-KIE。考克斯-1和考克斯-2对18：2n - 6的氧化作用通过[11，11-H-2（2）]-和[11 S-H-2]18：2n - 6的孵育进行探测，D-KIE为3-5。相比之下，考克斯-1和8 R-DOX对20：1 n- 6和18：1 n- 9烯丙基碳的更高能量需求的氢提取都伴随着大的D-KIE（>20）。(C)2012爱思唯尔有限公司版权所有。
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-H-2]18:2n - 6 with similar D-KIE (similar to 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-H-2(2)]- and [11S-H-2]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). (C) 2012 Elsevier B.V. All rights reserved.