Interactions between nitric oxide and peroxynitrite during prostaglandin endoperoxide H synthase-1 catalysis: a free radical mechanism of inactivation.
Peroxynitrite (ONOO(-)) can serve either as a peroxide substrate or as an inactivator of prostaglandin endoperoxide H synthase-1 (PGHS-1). Herein, the mechanism of PGHS-1 inactivation by ONOO(-) and the modulatory role that nitric oxide (*NO) plays in this process were studied. PGHS-1 reacted with ONOO(-) with a second-order rate constant of 1.7 x 10(7) M(-1) s(-1) at pH 7.0 and 8 degrees C. In the absence of substrates, the enzyme was dose-dependently inactivated by ONOO(-) in parallel with 3-nitrotyrosine formation. However, when PGHS-1 was incubated with ONOO(-) in the presence of substrates, the direct reaction with ONOO(-) was less relevant and ONOO(-)-derived radicals became involved in enzyme inactivation. Bicarbonate at physiologically relevant concentrations enhanced PGHS-1 inactivation and nitration by ONOO(-), further supporting a free radical mechanism. Importantly, *NO (0.4-1.5 microM min(-1)) was able to spare the peroxidase activity of PGHS-1 but it enhanced ONOO(-)-mediated inactivation of cyclooxygenase. The observed differential effects of *NO on ONOO(-)-mediated PGHS-1 inactivation emphasize a novel aspect of the complex modulatory role that *NO plays during inflammatory processes. We conclude that ONOO(-)-derived radicals inactivate both peroxidase and cyclooxygenase activities of PGHS-1 during enzyme turnover. Finally, our results reconcile the proposed alternative effects of ONOO(-) on PGHS-1 (activation versus inactivation).
Trostchansky
Andrés
A
Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay.
O'Donnell
Valerie B
VB
Goodwin
Douglas C
DC
Landino
Lisa M
LM
Marnett
Lawrence J
LJ
Radi
Rafael
R
Rubbo
Homero
H
D002384:Catalysis; D005609:Free Radicals; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000187:drug effects* / Q000378:metabolism; D013379:Substrate Specificity
D005609:Free Radicals; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D011451:Prostaglandin-Endoperoxide Synthases