Structural characterization of arachidonyl radicals formed by aspirin-treated prostaglandin H synthase-2

Ah Lim Tsai, Graham Palmer, Gang Wu, Sheng Peng, Nicole M. Okeley, Wilfred A. Van Der Donk, Richard J. Kulmacz

Research output: Contribution to journalArticlepeer-review


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 ASAPGHS-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, 13,15d2-AA, and octadeuterated AA (d8-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 16d2-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.

Original languageEnglish (US)
Pages (from-to)38311-38321
Number of pages11
JournalJournal of Biological Chemistry
Issue number41
StatePublished - Oct 11 2002
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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