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 microM. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Bakovic
M
M
Department of Chemistry, University of Alberta, Edmonton, Canada.
Dunford
H B
HB
D000818:Animals; D003373:Coumaric Acids / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011422:Propionates; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep
D003373:Coumaric Acids; D011422:Propionates; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D011451:Prostaglandin-Endoperoxide Synthases; C495469:p-coumaric acid