title: Novel insights into cyclooxygenases, linoleate diol synthases, and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs.
abstract: 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-(2)H]18:2n-6 with similar D-KIE (~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-(2)H(2)]- and [11S-(2)H]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).
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
authors.forename: Inga
authors.initials: I
authors.lastname: Hoffmann
authors.forename: Mats
authors.initials: M
authors.lastname: Hamberg
authors.forename: Roland
authors.initials: R
authors.lastname: Lindh
authors.forename: Ernst H
authors.initials: EH
authors.lastname: Oliw
mesh_terms: D055162:Biocatalysis; D002853:Chromatography, Liquid; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D058945:Lipoxygenases / Q000378:metabolism*; D013058:Mass Spectrometry; D008956:Models, Chemical; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D005231:Fatty Acids, Unsaturated; D003903:Deuterium; D010105:Oxygenases; D058945:Lipoxygenases; C122014:linoleate diol synthase; D011451:Prostaglandin-Endoperoxide Synthases; D010100:Oxygen