EPR investigation of the active site of recombinant human 5-lipoxygenase: inhibition by selenide.
Lipoxygenases are a group of non-heme iron dioxygenases which catalyze the formation of lipid hydroperoxides from unsaturated fatty acids. 5-Lipoxygenase (5LO) is of particular interest for formation of leukotrienes and lipoxins, implicated in inflammatory processes. In this study, electron paramagnetic resonance (EPR) spectroscopy was used to investigate the active site iron of purified recombinant human 5-lipoxygenase (5LO), and to explore the action of selenide on 5LO. After oxidation by lipid hydroperoxides, 5LO exhibited axial EPR spectra typified by a signal at g = 6.2. However, removal of the lipid hydroperoxides, their metabolites, and the solvent ethanol from the samples resulted in a shift to more rhombic EPR spectra (g = 5.17 and g = 9.0). Thus, many features of 5LO and soybean lipoxygenase-1 EPR spectra were similar, indicating similar flexible iron ligand arrangements in these lipoxygenases. Selenide (1.5 microM) showed a strong inhibitory effect on the enzyme activity of 5LO. In EPR, selenide abolished the signal at g = 6.2, typical for enzymatically active 5LO. Lipid hydroperoxide added to selenide-treated 5LO could not reinstate the signal at g = 6.2, indicating an irreversible change of the coordination of the active site iron.
Hammarberg
T
T
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. Tove.Hammarberg@dbb.su.se
Kuprin
S
S
Rådmark
O
O
Holmgren
A
A
D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000431:Ethanol / Q000737:chemistry; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008054:Lipid Peroxides / Q000737:chemistry; D016859:Lipoxygenase Inhibitors; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012643:Selenium / Q000737:chemistry*
D004791:Enzyme Inhibitors; D005290:Ferric Compounds; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; D011994:Recombinant Proteins; D000431:Ethanol; D007501:Iron; D001094:Arachidonate 5-Lipoxygenase; D012643:Selenium