title: Structural characterization of arachidonyl radicals formed by aspirin-treated prostaglandin H synthase-2.
abstract: Peroxide-generated tyrosyl radicals in both prostaglandin H synthase (PGHS) isozymes have been demonstrated to couple the peroxidase and cyclooxygenase activities by serving as the immediate oxidant for arachidonic acid (AA) in cyclooxygenase catalysis. Acetylation of Ser-530 of PGHS-1 by aspirin abolishes all oxygenase activity and transforms the peroxide-induced tyrosyl radical from a functional 33-35-gauss (G) wide doublet/wide singlet to a 26-G narrow singlet unable to oxidize AA. In contrast, aspirin-treated PGHS-2 (ASA-PGHS-2) no longer forms prostaglandins but retains oxygenase activity forming 11(R)- and 15(R)-hydroperoxyeicosatetraenoic acid and also retains the EPR line-shape of the native peroxide-induced 29-30-G wide singlet radical. To evaluate the functional role of the wide singlet radical in ASA-PGHS-2, we have examined the ability of this radical to oxidize AA in single-turnover EPR studies. Anaerobic addition of AA to ASA-PGHS-2 immediately after formation of the wide singlet radical generated either a 7-line EPR signal similar to the pentadienyl AA radical obtained in native PGHS-2 or a 26-28-G singlet radical. These EPR signals could be accounted for by a pentadienyl radical and a strained allyl radical, respectively. Experiments using 11d-AA, 13(R)d-AA, 15d-AA, 13,15d(2)-AA, and octadeuterated AA (d(8)-AA) confirmed that the unpaired electron in the pentadienyl radical is delocalized over C11, C13, and C15. A 6-line EPR radical was observed when 16d(2)-AA was used, indicating only one strongly interacting C16 hydrogen. These results support a functional role for peroxide-generated tyrosyl radicals in lipoxygenase catalysis by ASA-PGHS-2 and also indicate that the AA radical in ASA-PGHS-2 is more constrained than the corresponding radical in native PGHS-2.
authors.affiliation: Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA. ah-lim.tsai@uth.tmc.edu
authors.forename: Ah-lim
authors.initials: AL
authors.lastname: Tsai
authors.forename: Graham
authors.initials: G
authors.lastname: Palmer
authors.forename: Gang
authors.initials: G
authors.lastname: Wu
authors.forename: Sheng
authors.initials: S
authors.lastname: Peng
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Okeley
authors.forename: Wilfred A
authors.initials: WA
authors.lastname: van der Donk
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Kulmacz
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000737:chemistry* / Q000378:metabolism; D001241:Aspirin / Q000737:chemistry*; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008280:Magnetics; D008565:Membrane Proteins; D015394:Molecular Structure; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000378:metabolism; D012756:Sheep; D014443:Tyrosine / Q000737:chemistry
chemical_list: D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D005609:Free Radicals; D007527:Isoenzymes; D008565:Membrane Proteins; D016718:Arachidonic Acid; D014443:Tyrosine; D006861:Hydrogen Peroxide; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D001241:Aspirin