Abstract
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 × 10 7 M − 1 s − 1 at pH 7.0 and 8 °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 μM 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).
Published Version
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