Abstract
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.
Highlights
Prostaglandin H synthase (PGHS)l plays a key role in controlling the biosynthesis of various physiologically important prostaglandins (1)
PGHS-l has two enzyme functions: a cyclooxygenase, which converts arachidonic acid to PGG2, and a peroxidase, which catalyzes the transformation of PGG2 to PGH2 (1-3)
Control Reactions of PGHS-l with Arachidonate or Hydroperoxide-Even at 0 °C, the relatively concentrated solutions of PGHS-1 used for the EPR experiments react quantitatively with arachidonate in air within a few seconds, producing a large tyrosyl radical EPR signal (8)
Summary
Vol 270, No 18, Issue of May 5, pp. 10503-10508, 1995 Printed in U,S.A. Spectroscopic Evidence for Reaction of Prostaglandin H Synthase-l Tyrosyl Radical with Arachidonic Acid*. 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-l and support the proposed role for a tyrosyl radical in cyclooxygenase catalysis. In the studies described here, we have used single turnover experiments to demonstrate that the peroxidase-associated WD radical reacts with arachidonic acid to form a carbon-centered radical, providing direct evidence for a key step in the proposed tyrosyl radical mechanism
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