Institute for Medical Physics and Biophysics, Medical Faculty, Leipzig University, Leipzig, Germany; Translational Center for Regenerative Medicine Leipzig, Leipzig University, Leipzig, Germany.
Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany; LIFE-Leipzig Research Center for Civilization Diseases, Universität Leipzig, Leipzig, Germany.
Free Radic Biol Med. 2015 Aug;85:148-56. doi: 10.1016/j.freeradbiomed.2015.04.015. Epub 2015 Apr 22.
Human 5-lipoxygenase (5-LOX) oxidizes arachidonic acid to 5S-hydroperoxy-6 E,8 Z,11 Z,14 Z-eicosatetraenoic acid (5-HpETE) and leukotriene (LT) A4. In neutrophils, LTA4 is further converted to the potent chemoattractant LTB4. These cells also contain the heme enzyme myeloperoxidase (MPO), which produces several potent oxidants such as hypochlorous acid (HOCl), which are involved in pathogen defense and immune regulation. Here, we addressed the question whether MPO-derived oxidants are able to affect the activity of 5-LOX and the product profile of this enzyme. Human 5-LOX was incubated with increasing amounts of HOCl or HOBr. Afterward, arachidonic acid metabolites of 5-LOX were analyzed by reverse-phase high-performance liquid chromatography as well as by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of 5-LOX with the MPO-derived oxidants significantly changed the product profile of 5-LOX. Thereby, HOCl and HOBr increased the ratio of 5-H(p)ETE to 6-trans-LTB4 in a concentration-dependent manner. At low oxidant concentrations, there was a strong decrease in the yield of 6-trans-LTB4, whereas 5-HpETE did not change or increased. Additionally, the formation of 8-HpETE and 12-HpETE by 5-LOX rose slightly with increasing HOCl and HOBr. Comparable results were obtained with the MPO-H2O2-Cl(-) system when glucose oxidase and glucose were applied as a source of H2O2. This was necessary because of a strong impairment of 5-LOX activity by H2O2. In summary, MPO-derived oxidants showed a considerable impact on 5-LOX, impairing the epoxidation of 5-HpETE, whereas the hydroperoxidation of arachidonic acid was unaffected. Apparently, this was caused by an oxidative modification of critical amino acid residues of 5-LOX. Further work is necessary to assess the specific type and position of oxidation in the substrate-binding cavity of 5-LOX and to specify whether this interaction between 5-LOX and MPO-derived oxidants also takes place in stimulated neutrophils.
人 5-脂氧合酶（5-LOX）将花生四烯酸氧化为 5S-过氧-6 E，8 Z，11 Z，14 Z-二十碳四烯酸（5-HpETE）和白三烯（LT）A4。在中性粒细胞中，LTA4 进一步转化为有效的趋化因子 LTB4。这些细胞还含有血红素酶髓过氧化物酶（MPO），它产生几种强氧化剂，如次氯酸（HOCl），参与病原体防御和免疫调节。在这里，我们研究了 MPO 衍生的氧化剂是否能够影响 5-LOX 的活性及其产物谱。用递增浓度的 HOCl 或 HOBr 孵育人 5-LOX。之后，通过反相高效液相色谱法以及液相色谱-电喷雾串联质谱法分析 5-LOX 的花生四烯酸代谢物。MPO 衍生的氧化剂孵育显著改变了 5-LOX 的产物谱。HOCl 和 HOBr 以浓度依赖性方式增加 5-H（p）ETE 与 6-反式-LTB4 的比例。在低氧化剂浓度下，6-反式-LTB4 的产率明显下降，而 5-HpETE 不变或增加。此外，5-LOX 形成的 8-HpETE 和 12-HpETE 略有增加随着 HOCl 和 HOBr 的增加而增加。当应用葡萄糖氧化酶和葡萄糖作为 H2O2 的来源时，MPO-H2O2-Cl（-）系统获得了类似的结果。这是必要的，因为 H2O2 对 5-LOX 活性有很强的抑制作用。总之，MPO 衍生的氧化剂对 5-LOX 有显著影响，损害 5-HpETE 的环氧化，而对花生四烯酸的过氧水解没有影响。显然，这是由于 5-LOX 关键氨基酸残基的氧化修饰所致。需要进一步研究以评估 5-LOX 底物结合腔中氧化的具体类型和位置，并确定 5-LOX 和 MPO 衍生的氧化剂之间的这种相互作用是否也发生在受刺激的中性粒细胞中。
