Schwarz K, Borngräber S, Anton M, Kuhn H
Institute of Biochemistry, University Clinics (Charité), Humboldt University, Berlin, Germany.
Biochemistry. 1998 Nov 3;37(44):15327-35. doi: 10.1021/bi9816204.
For oxygenation of polyenoic fatty acids by 12- and 15-lipoxygenases the methyl terminus of the substrate constitutes the signal for the initial hydrogen abstraction. In contrast, for 5-lipoxygenases an inverse head to tail substrate orientation has been proposed. However, recent structure-based sequence alignments suggested a conserved uniform substrate orientation for 5S- and 15S-lipoxygenation. Oxygenation of 15S-HETE derivatives by various wild-type and mutant lipoxygenases was investigated, and the evidence proved an inverse substrate orientation: (i) Substrate affinity and Vmax of 15S-HETE oxygenation by arachidonic acid 15-lipoxygenases are >1 order of magnitude lower than the corresponding data for polyenoic fatty acids. 5S,15S- and 14R, 15S-DiH(P)ETE were identified as major reaction products. (ii) Methylation of the carboxylate group of 15S-HETE augmented the reaction rate and shifted the reaction specificity strongly toward 5S-lipoxygenation. In contrast, methyl arachidonate was less effectively oxygenated than the free acid. Methylation of 15S-HETrE(8,11,14), which lacks the C5-C6 double bond, was without major impact on the oxygenation rate and on the product specificity. (iii) Introduction of a bulky glycerol moiety at the carboxylic group of 15S-HETE reversed the kinetic effects of methylation and led to a 14R-oxygenation of the substrate. (iv) When the product pattern of 15S-HETE oxygenation by the recombinant wild-type rabbit 15-lipoxygenase was compared with that formed by the Arg403Leu mutant, 5S- and 8S-lipoxygenations were augmented and 14R, 15S-DiH(P)ETE formation was impaired. (v) Phe353Leu or Ile418Ala mutation of the same enzyme, which favored 12S-HETE formation from arachidonic acid, strongly augmented 8S-lipoxygenation of 15S-HETE methyl ester. These kinetic data and the alterations in the product specificity are consistent with the concept of an inverse head to tail substrate orientation during the oxygenation of 15S-HETE methyl ester and/or of free 15S-HETE by 15-LOXs. For 5S- and 8S-lipoxygenation, 15-HETE may slide into the substrate binding pocket with its carboxy terminus approaching the doubly allylic methylenes C-7 or C-10 to the non-heme iron.
对于12-脂氧合酶和15-脂氧合酶对多不饱和脂肪酸的氧化作用，底物的甲基末端构成了初始氢提取的信号。相比之下，对于5-脂氧合酶，有人提出底物呈头尾相反的取向。然而，最近基于结构的序列比对表明，5S-和15S-脂氧合作用存在保守的统一底物取向。研究了各种野生型和突变型脂氧合酶对15S-HETE衍生物的氧化作用，证据证明底物取向相反：（i）花生四烯酸15-脂氧合酶对15S-HETE的氧化作用的底物亲和力和Vmax比多不饱和脂肪酸的相应数据低1个数量级以上。5S,15S-和14R,15S-二氢（磷）二十碳四烯酸被鉴定为主要反应产物。（ii）15S-HETE羧基的甲基化提高了反应速率，并使反应特异性强烈转向5S-脂氧合作用。相比之下，花生四烯酸甲酯的氧化效率低于游离酸。缺乏C5-C6双键的15S-HETrE(8,11,14)的甲基化对氧化速率和产物特异性没有重大影响。（iii）在15S-HETE的羧基引入一个庞大的甘油部分，逆转了甲基化的动力学效应，并导致底物的14R-氧化。（iv）将重组野生型兔15-脂氧合酶对15S-HETE的氧化产物模式与Arg403Leu突变体形成的产物模式进行比较时，5S-和8S-脂氧合作用增强，14R,15S-二氢（磷）二十碳四烯酸的形成受到损害。（v）同一酶的Phe353Leu或Ile418Ala突变有利于从花生四烯酸形成12S-HETE，强烈增强了15S-HETE甲酯的8S-脂氧合作用。这些动力学数据和产物特异性的改变与15S-HETE甲酯和/或游离15S-HETE被15-脂氧合酶氧化过程中头尾相反的底物取向概念一致。对于5S-和8S-脂氧合作用，15-HETE可能以其羧基末端接近非血红素铁的双烯丙基亚甲基C-7或C-10的方式滑入底物结合口袋。
