Analysis of hydroperoxide-induced tyrosyl radicals and lipoxygenase activity in aspirin-treated human prostaglandin H synthase-2.
A hydroperoxide-induced tyrosyl radical has been proposed as a key cyclooxygenase intermediate for the "basal" isoform of prostaglandin H synthase (PGHS-1). In the present study with the "inducible" isoform (PGHS-2), hydroperoxide was also found to generate a radical in high yield, a wide singlet at g = 2.0058 (29 G peak to trough). Reaction of PGHS-2 with a tyrosine-modifying reagent, tetranitromethane (TNM), resulted in cyclooxygenase inactivation and a much narrower radical EPR signal (22 G peak to trough). Addition of a cyclooxygenase inhibitor, nimesulide, similarly resulted in a narrow PGHS-2 radical. In PGHS-1, cyclooxygenase inhibition by tyrosine nitration with TNM or by active site ligands leads to generation of a narrow EPR instead of a wide EPR, with both signals originating from authentic tyrosyl radicals, indicating that the hydroperoxide-induced radicals in PGHS-2 are also tyrosyl radicals. Treatment of PGHS-2 with aspirin (acetyl salicylic acid, ASA) was previously shown to result in acetylation of a specific serine residue, cyclooxygenase inhibition, and increased lipoxygenase activity. Acetylation of PGHS-1 by ASA, in contrast, inhibited both lipoxygenase and cyclooxygenase activity. We now have found the ASA-treated PGHS-2 radical to be indistinguishable from that in control PGHS-2. Addition of nimesulide to ASA-treated PGHS-2 inhibited the lipoxygenase and resulted in a narrow radical EPR like that seen in PGHS-2 treated with TNM or nimesulide alone. Retention of PGHS-2 oxygenase activity was thus associated with retention of the native radical, and loss of activity was associated with alteration of the radical. Both native and ASA-treated PGHS-2 produced only the R stereoisomer of 11- and 15-HETE, demonstrating that the lipoxygenase stereochemistry was not changed by ASA. Native and ASA-treated PGHS-2 had lipoxygenase K(m) values considerably higher than that of the control PGHS-2 cyclooxygenase. Taken together, these results suggest that the same PGHS-2 tyrosyl radical serves as the oxidant for both cyclooxygenase and lipoxygenase catalysis and that acetylation of PGHS-2 by ASA favors arachidonate binding in an altered conformation which results in abstraction of the pro-R hydrogen from C13 and formation of 11(R)- and 15(R)-HETE.
Xiao
G
G
Department of Internal Medicine, University of Texas Health Science Center at Houston 77030, USA.
Tsai
A L
AL
Palmer
G
G
Boyar
W C
WC
Marshall
P J
PJ
Kulmacz
R J
RJ
D016718:Arachidonic Acid / Q000378:metabolism; D001241:Aspirin / Q000494:pharmacology*; D051546:Cyclooxygenase 2; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D005609:Free Radicals / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007527:Isoenzymes / Q000737:chemistry* / Q000187:drug effects* / Q000378:metabolism; D007700:Kinetics; D008084:Lipoxygenase / Q000378:metabolism*; D008565:Membrane Proteins; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000187:drug effects* / Q000378:metabolism; D013237:Stereoisomerism; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000187:drug effects*
D005609:Free Radicals; D007527:Isoenzymes; D008565:Membrane Proteins; D016718:Arachidonic Acid; D014443:Tyrosine; D006861:Hydrogen Peroxide; D008084:Lipoxygenase; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D013774:Tetranitromethane; D001241:Aspirin