title: Eosinophil 15-lipoxygenase is a leukotriene A4 synthase.
abstract: 5-Lipoxygenase is the first committed enzyme in the leukotriene biosynthetic pathway and is known to catalyze not only the first oxygenation of arachidonate to form 5(S)-hydroperoxyeicosatetraenoic acid (5(S)-HPETE), but also dehydration of this intermediate into leukotriene A4 (LTA4) by an activity termed leukotriene A4 synthase. Inhibition of cytosolic 5-lipoxygenase prepared from human blood granulocytes with zileuton (100 microM) was virtually complete, but LTA4 synthase activity was only inhibited by 47%. Structural characterization of eicosanoids synthesized in these preparations revealed an abundance of 15-lipoxygenase metabolites including 15-HETE when arachidonate was used as substrate and 5(S),15(S)-dihydroxy-6,8,11,13(E,E,Z,Z)-eicosatetraenoic acid when 5(S)-HPETE was used as substrate. When neutrophils were prepared that contained less than 1% eosinophil contamination, zileuton was found to almost completely inhibit all 5-lipoxygenase, as well as LTA4 synthase products. Immunochemical analysis of the supernatants from purified neutrophils and eosinophils confirmed the previous observation that neutrophils do not express 15-lipoxygenase. Incubation of 5(S)-HPETE with recombinant mammalian 15-lipoxygenase resulted in the formation of 6-trans-LTB4 and 6-trans-12-epi-LTB4 as LTA4 products, as well as the 12-lipoxygenase product 5(S),12(S)-diHPETE. The mechanism of action of 15-lipoxygenase acting as an LTA4 synthase is proposed to involve removing the pro-R hydrogen atom at carbon-10 of 5(S)-HPETE, which is antarafacial to the hydroperoxy group to yield LTA4.
authors.affiliation: National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
authors.forename: D K
authors.initials: DK
authors.lastname: MacMillan
authors.forename: E
authors.initials: E
authors.lastname: Hill
authors.forename: A
authors.initials: A
authors.lastname: Sala
authors.forename: E
authors.initials: E
authors.lastname: Sigal
authors.forename: T
authors.initials: T
authors.lastname: Shuman
authors.forename: P M
authors.initials: PM
authors.lastname: Henson
authors.forename: R C
authors.initials: RC
authors.lastname: Murphy
mesh_terms: D001093:Arachidonate 15-Lipoxygenase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D001094:Arachidonate 5-Lipoxygenase / Q000145:classification / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D002474:Cell-Free System; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004804:Eosinophils / Q000201:enzymology / Q000378:metabolism; D006098:Granulocytes / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis; D006918:Hydroxyurea / Q000031:analogs & derivatives / Q000494:pharmacology; D017572:Leukotriene A4 / Q000096:biosynthesis*; D015289:Leukotrienes / Q000378:metabolism; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D008956:Models, Chemical; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D006893:Hydroxyeicosatetraenoic Acids; D017572:Leukotriene A4; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; D011994:Recombinant Proteins; D016718:Arachidonic Acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; C025348:arachidonic acid 5-hydroperoxide; D001093:Arachidonate 15-Lipoxygenase; D001094:Arachidonate 5-Lipoxygenase; C063449:zileuton; D006918:Hydroxyurea