Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis.

Abstract

High levels of NaCN (20 to 250 mM) were required to inhibit cyclooxygenase catalysis and cause extended lag periods (up to 1.6 min), whereas CO failed to inhibit catalysis. This NaCN inhibition was easily overcome by endogenous or exogenous hydroperoxides. Added hydroperoxides acted to eliminate lag periods without undergoing net conversion to other chemical species. In addition, experiments with glutathione peroxidase inhibition showed that hydroperoxides were essential not only in the early phases, but throughout catalysis. In spectrophotometric experiments, NaCN formed a complex with ferriheme cyclooxygenase (Kd = 1.3 mM) and inhibited hydroperoxide interaction with this form of the enzyme. Phenolic antioxidants, only slightly extended lag periods while inhibiting oxygenation rates more than 50%. Low levels of phenol (which is normally stimulatory) or alpha-naphthol when combined with NaCN or glutathione peroxidase (agents which interfere with peroxide activation) resulted in potent synergistic inhibition with long lag times. A mechanism consistent with all of the above properties of cyclooxygenase has been elucidated, Further mechanistic explanation was sought for reaction-catalyzed self-inactivation of cyclooxygenase. This phenomenon could not be explained simply by heme lability, or cyclooxygenase sensitivity to destruction by ambient hydroperoxides, Rather, it appears to involve a destructive reaction intermediate intrinsic to involve a destructive reaction intermediate intrinsic to the cyclooxygenase mechanism.