Reactions of prostaglandin endoperoxide synthase with hydroperoxide and reducing substrates under single turnover conditions.

The peroxidase reaction of prostaglandin endoperoxide synthase was investigated by transient state kinetics using stoichiometric amounts of substrates. The rate constants for the conversion of compound I to intermediate II determined with a stoichiometric amount of hydroperoxide were found to be lower by an order of magnitude than when an excess of hydroperoxide was used. The difference was attributed to ability of the compound I of prostaglandin endoperoxide synthase to be reduced by the excess of hydroperoxide. This suggests that the true rate constant of unimolecular conversion compound I to intermediate II at 3 degrees C is 5-10 s-1 instead of 50-200 s-1 as reported before. The latter value rather characterizes the combined process of spontaneous and hydroperoxide-dependent transformation of compound I. Stoichiometric amounts of reducing substrates significantly stimulated transformation of compound I. This effect could not be entirely explained by their reducing action, which was measured by following the oxidation kinetics. The results of the global fit of the experimental data suggest that reducing substrates, in addition to their direct action in reducing compound I to compound II, indirectly stimulate transformation of compound I to the tyrosyl radical form of intermediate II, thereby stimulating the cyclooxygenase reaction.