INTIMATE RELATION BETWEEN CYCLOOXYGENASE AND PEROXIDASE-ACTIVITIES OFPROSTAGLANDIN-H SYNTHASE - PEROXIDASE REACTION OF FERULIC ACID AND ITS INFLUENCE ON THE REACTION OF ARACHIDONIC-ACID

The oxidation of ferulic acid by hydrogen peroxide catalyzed by prosta glandin 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, determine d from steady-state results, is (1.31 +/- 0.1) X 10(6) M(-1) s(-1), an d for the reaction of prostaglandin H synthase-compound II with feruli c acid it is (5.5 +/- 0.3) X 10(6) M(-1) s(-1). Cyclooxygenase and per oxidase functions of prostaglandlin H synthase were studied by compari ng the initial rates of reaction of the cyclooxygenase substrate, arac hidonic acid, and a peroxidase reducing substrate. ferulic acid, in mi xtures of the two substrates. For both an equimolar ratio of arachidon ic and ferulic acids and ferulic acid in excess of arachidonic acid a stimulation of the cyclooxygenase reaction is observed. The concentrat ion of ferulic acid necessary to produce 50% stimulation of 0.2 mM ara chidonic acid oxidation is 0.14 +/- 0.02 mM. A striking feature of our results is that prostaglandin H synthase catalyzes oxidation of the t wo substrates in a constant and fixed molar ratio of ferulic acid to a rachidonic acid of 2:1, despite widely different starting concenration s. If arachidonic acid is in excess of ferulic acid, enzyme inactivati on occurs. The results can be explained by an interconnected cyclooxyg enase-peroxidase unbranched free radical mechanism in which arachidoni c acid reacts with either the ferryl oxygen or the porphyrin pi-cation radical part of a conventional peroxidase compound I (a Fe-IV=O porph yrin pi-cation radical) and ferulic acid reacts with compound II (Fe-I V=0). The ferulic acid also acts as a hydrogen atom donor to the hydro peroxyl radical, PG(G2). or alternatively to the tyrosyl radical provi ding protection from inactivation. Our data provide strong evidence ag ainst a cyclooxygenase branched chain free radical mechanism, in which a cyclooxygenase cycle, once started, can operate with no connection to the peroxidase cycle.