title: Identification of Tyr504 as an alternative tyrosyl radical site in human prostaglandin H synthase-2.
abstract: Hydroperoxides induce formation of a tyrosyl radical on Tyr385 in prostaglandin H synthase (PGHS). The Tyr385 radical initiates hydrogen abstraction from arachidonic acid, thereby mechanistically connecting the peroxidase and cyclooxygenase activities. In both PGHS isoforms the tyrosyl radical undergoes a time-dependent transition from a wide doublet to a wide singlet species; pretreatment with cyclooxygenase inhibitors results in a third type of signal, a narrow singlet [Tsai, A.-L.; Kulmacz, R. J. (2000) Prost. Lipid Med. 62, 231-254]. These transitions have been interpreted as resulting from Tyr385 ring rotation, but could also be due to radical migration from Tyr385 to another tyrosine residue. PATHWAYS analysis of PGHS crystal structures identified four tyrosine residues with favorable predicted electronic coupling: residues 148, 348, 404, and 504 (ovine PGHS-1 numbering). We expressed recombinant PGHS-2 proteins containing single Tyr --> Phe mutations at the target residues, a quadruple mutant with all four tyrosines mutated, and a quintuple mutant, which also contains a Y385F mutation. All mutants bind heme and display appreciable peroxidase activity, and with the exception of the quintuple mutant, all retain cyclooxygenase activity, indicating that neither of the active sites is significantly perturbed. Reaction of the Y148F, Y348F, and Y404F mutants with EtOOH generates a wide singlet EPR signal similar to that of native PGHS-2. However, reaction of the Y504F and the quadruple mutants with peroxide yields persistent wide doublets, and the quintuple mutant is EPR silent. Nimesulide pretreatment of Y504F and the quadruple mutant results in an abnormally small amount of wide doublet signal, with no narrow singlet being formed. Therefore, the formation of an alternative tyrosine radical on Tyr504 probably accounts for the transition from a wide doublet to a wide singlet in native PGHS-2 and for formation of a narrow singlet in complexes of PGHS-2 with cyclooxygenase inhibitors.
authors.affiliation: Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
authors.forename: Corina E
authors.initials: CE
authors.lastname: Rogge
authors.forename: Wen
authors.initials: W
authors.lastname: Liu
authors.forename: Gang
authors.initials: G
authors.lastname: Wu
authors.forename: Lee-Ho
authors.initials: LH
authors.lastname: Wang
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Kulmacz
authors.forename: Ah-Lim
authors.initials: AL
authors.lastname: Tsai
mesh_terms: D001667:Binding, Competitive / Q000235:genetics; D002384:Catalysis; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005609:Free Radicals / Q000737:chemistry*; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013449:Sulfonamides / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
chemical_list: D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D005609:Free Radicals; D007527:Isoenzymes; D008565:Membrane Proteins; D011994:Recombinant Proteins; D013449:Sulfonamides; C408694:tyrosine radical; D014443:Tyrosine; D010544:Peroxidases; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; C012655:nimesulide