KINETICS OF THE OXIDATION OF P-COUMARIC ACID BY PROSTAGLANDIN-H SYNTHASE AND HYDROGEN-PEROXIDE

Steady-state kinetics of the oxidation of p-coumaric acid (CA) by prostaglandin H synthase and hydrogen peroxide was studied at 25-degrees-C in 0.1 M phosphate buffer, pH 8.0, using a stopped-flow apparatus. The following evidence supports a mechanism in which CA serves as a reducing substrate for prostaglandin H synthase through two one-electron oxidation steps: (a) the oxidation product of CA is the same in the prostaglandin H synthase/hydrogen peroxide and the horseradish peroxidase/hydrogen peroxide systems; (b) an identical steady-state enzyme intermediate (compound II) is present in both systems; (c) CA stimulates the cyclooxygenase activity of prostaglandin H synthase; the concentration of CA that produces 50% stimulation, A50, is 350 +/- 30 muM. On the time scale of our experiments, the inactivation of prostaglandin H synthase by hydrogen peroxide was insignificant when CA was present. A molar absorptivity of 17.2 +/- 0.9 mM-1 cm-1 at 300 nm was determined for CA which was used to follow the initial rate of disappearance of CA. The reaction of CA with hydrogen peroxide catalyzed by prostaglandin H synthase showed saturation behavior. An irreversible reaction mechanism for the steady-state kinetics of prostaglandin H synthase is proposed which is consistent with all of our experimental results. Under steady-state conditions, the second-order rate constants for the reactions of prostaglandin H synthase with hydrogen peroxide and prostaglandin H synthase-compound II with CA are (9.2 +/- 0.1) X 10(5) and (2.5 +/- 0.1) X 10(6) M-1 s-1, respectively. Steady-state kinetic studies of prostaglandin H synthase can be performed by using stopped-flow spectrophotometry (dead time 1-5 ms) with considerable advantage over conventional spectrophotometry (dead time of the order of 1-5 s), in particular with regard to avoiding problems of enzyme inactivation.