Spectroscopic evidence for reaction of prostaglandin H synthase-1 tyrosyl radical with arachidonic acid.
The coupling between the peroxidase and cyclooxygenase activities of prostaglandin H synthase (PGHS) has been proposed to be mediated by a critical tyrosyl radical through a branched chain mechanism (Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 321-328). In this study, we have examined the ability of PGHS isoform-1 (PGHS-1) tyrosyl radicals to react with arachidonate. Anaerobic addition of arachidonate following formation of the peroxide-induced wide doublet or wide singlet tyrosyl radical led to disappearance of the tyrosyl radicals and emergence of a new EPR signal, which is distinct from known PGHS-1 tyrosyl radicals. The new radical was clearly derived from arachidonate because its EPR line shape changed when 5,6,8,9,11,12,14,15-octadeuterated arachidonate was used. Subsequent addition of oxygen to samples containing the fatty acyl radical resulted in regeneration of tyrosyl radical EPR. In contrast, the peroxide-generated tyrosyl radical in indomethacin-treated PGHS-1 (a narrow singlet) failed to react with arachidonate, consistent with the cyclooxygenase inhibition by indomethacin. These results indicate that the peroxide-generated wide doublet and wide singlet tyrosyl radicals serve as immediate oxidants of arachidonate bound at the cyclooxygenase active site to form a carbon-centered fatty acyl radical, which reacts with oxygen to form a hydroperoxide. These observations represent the first direct evidence of chemical coupling between the peroxidase reaction and arachidonate oxygenation in PGHS-1 and support the proposed role for a tyrosyl radical in cyclooxygenase catalysis.
Tsai
A
A
Department of Internal Medicine, University of Texas Health Science Center at Houston 77030, USA.
Kulmacz
R J
RJ
Palmer
G
G
D000818:Animals; D016718:Arachidonic Acid / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000737:chemistry; D007213:Indomethacin / Q000494:pharmacology; D008297:Male; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry*; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep; D014443:Tyrosine / Q000737:chemistry
D005609:Free Radicals; D016718:Arachidonic Acid; D014443:Tyrosine; D006861:Hydrogen Peroxide; D011451:Prostaglandin-Endoperoxide Synthases; D007213:Indomethacin