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

The peroxidase reaction of prostaglandin endoperoxide synthase was investig ated by transient state kinetics using stoichiometric amounts of substrates . The rate constants for the conversion of compound I to intermediate II de termined with a stoichiometric amount of hydroperoxide were found to be low er 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 s uggests that the true rate constant of unimolecular conversion compound I t o intermediate II at 3 degrees C is 5-10 s(-1) instead of 50-200 s(-1) as r eported before. The latter value rather characterizes the combined precess of spontaneous and hydroperoxide-dependent transformation of compound I. St oichiometric amounts of reducing substrates significantly stimulated transf ormation of compound I. This effect could not be entirely explained by thei r reducing action, which was measured by following the oxidation kinetics. The results of the global fit of the experimental data suggest that reducin g substrates, in addition to their direct action in reducing compound I to compound II, indirectly stimulate transformation of compound I to the tyros yl radical form of intermediate II, thereby stimulating the cyclooxygenase reaction. (C) 1999 Elsevier Science B.V. All rights reserved.