The oxidation of ferulic acid by hydrogen peroxide catalyzed by prostaglandin H synthase follows a modified ping-pong irreversible mechanism, as is the case for classical peroxidases. The rate constant for the reaction of prostaglandin H synthase with hydrogen peroxide, determined from steady-state results, is (1.31 +/- 0.1) X 10(6) M(-1) s(-1), and for the reaction of prostaglandin H synthase-compound II with ferulic acid it is (5.5 +/- 0.3) X 10(6) M(-1) s(-1). Cyclooxygenase and peroxidase functions of prostaglandlin H synthase were studied by comparing the initial rates of reaction of the cyclooxygenase substrate, arachidonic acid, and a peroxidase reducing substrate. ferulic acid, in mixtures of the two substrates. For both an equimolar ratio of arachidonic and ferulic acids and ferulic acid in excess of arachidonic acid a stimulation of the cyclooxygenase reaction is observed. The concentration of ferulic acid necessary to produce 50% stimulation of 0.2 mM arachidonic acid oxidation is 0.14 +/- 0.02 mM. A striking feature of our results is that prostaglandin H synthase catalyzes oxidation of the two substrates in a constant and fixed molar ratio of ferulic acid to arachidonic acid of 2:1, despite widely different starting concenrations. If arachidonic acid is in excess of ferulic acid, enzyme inactivation occurs. The results can be explained by an interconnected cyclooxygenase-peroxidase unbranched free radical mechanism in which arachidonic acid reacts with either the ferryl oxygen or the porphyrin pi-cation radical part of a conventional peroxidase compound I (a Fe-IV=O porphyrin pi-cation radical) and ferulic acid reacts with compound II (Fe-IV=0). The ferulic acid also acts as a hydrogen atom donor to the hydroperoxyl radical, PG(G2). or alternatively to the tyrosyl radical providing protection from inactivation. Our data provide strong evidence against a cyclooxygenase branched chain free radical mechanism, in which a cyclooxygenase cycle, once started, can operate with no connection to the peroxidase cycle.
