Kinetics of the reaction of prostaglandin H synthase compound II with ascorbic acid

The reduction of prostaglandin H synthase compound II by ascorbic acid in the presence of diethyldithiocarbamate was studied in 0.1 M phosphate buffer (pH 8.0) at 4.0 +/- 0.5 degrees C, by rapid scan spectrometry and transient state kinetics. A saturation effect and nonzero intercept were observed in the plot of pseudo-first-order rate constant versus ascorbic acid concentration. The saturation behavior suggests formation of a complex between prostaglandin H synthase compound II and ascorbic acid, whereas the nonzero intercept is attributable to the reaction of compound II of prostaglandin H synthase with diethyldithiocarbamate present in the system as a stabilizing agent. A rate equation has been derived which includes all pathways for the conversion of prostaglandin H synthase compound II back to native enzyme. Kinetic parameters for the reduction of compound II by ascorbic acid were obtained. They are the second-order rate constant of (1.4 +/- 0.5) X 10(5) M-1, S-1, for the formation of the compound II-ascorbic acid complex, the first-order rate constant of (14 +/- 4) S-1 for the oxidation-reduction reaction of the complex and its dissociation, and a parameter, Km of 92 +/- 10 microM analogous to the Michaelis-Menten constant. Thus we demonstrate that a quantitative kinetic study on the prostaglandin H synthase reactions can be performed in the presence of diethyldithiocarbamate.