Analysis of hydroperoxide-induced tyrosyl radicals and lipoxygenase activity in aspirin-treated human prostaglandin H synthase-2

Biochemistry. 1997 Feb 18;36(7):1836-45. doi: 10.1021/bi962476u.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Arachidonic Acid / metabolism
  • Aspirin / pharmacology*
  • Cyclooxygenase 2
  • Electron Spin Resonance Spectroscopy
  • Enzyme Activation / drug effects
  • Free Radicals / chemistry
  • Humans
  • Hydrogen Peroxide / pharmacology*
  • Isoenzymes / chemistry*
  • Isoenzymes / drug effects*
  • Isoenzymes / metabolism
  • Kinetics
  • Lipoxygenase / metabolism*
  • Membrane Proteins
  • Prostaglandin-Endoperoxide Synthases / chemistry*
  • Prostaglandin-Endoperoxide Synthases / drug effects*
  • Prostaglandin-Endoperoxide Synthases / metabolism
  • Stereoisomerism
  • Tetranitromethane / pharmacology
  • Tyrosine / analogs & derivatives*
  • Tyrosine / chemistry
  • Tyrosine / drug effects*


  • Free Radicals
  • Isoenzymes
  • Membrane Proteins
  • Arachidonic Acid
  • Tyrosine
  • Hydrogen Peroxide
  • Lipoxygenase
  • Cyclooxygenase 2
  • PTGS2 protein, human
  • Prostaglandin-Endoperoxide Synthases
  • Tetranitromethane
  • Aspirin