The reactions of native prostaglandin endoperoxide synthase with structurally different hydroperoxides have been investigated by using kinetic spectrophotometric scan and conventional and sequential mixing stopped-flow experiments. The second order rate constants for compound I formation are (5.9 +/- 0.1) x 10(4) M(-1) s(-1) using t-butyl hydroperoxide as the oxidant, (2.5 +/- 0.1) x 10(6) M(-1) s(-1) for ethyl hydroperoxide and (5.1 +/- 0.6) x 10(7) M(-1) s(-1) for m-chloroperoxybenzoic acid at pH 7.0, 6.7 +/- 0.2 degrees C, and ionic strength 0.1 M. Sequential mixing, transient state experiments show for the first time that all hydroperoxides reduce compound I in a bimolecular reaction. Ethyl hydroperoxide, t-butyl hydroperoxide, and m-chloroperoxybenzoic acid react directly with compound I. The natural substrate prostaglandin G(2) forms a transient complex with compound I before the reduction step occurs. Therefore, compound I initially transforms to compound II, not to the compound I-tyrosyl radical. Second order rate constants for the reactions of compound I are (2.9 +/- 0.2) x 10(4) for t-butyl hydroperoxide, (3.5 +/- 0.5) x 10(4) for hydrogen peroxide, (4.2 +/- 0.2) x 10(4) for ethyl hydroperoxide, and (4.2 +/- 0.3) x 10(5) for m-chloroperoxybenzoic acid, all in units of M(-1) s(-1) and same conditions as for compound I formation. The rate of reaction of prostaglandin G(2) with compound I, calculated from the ratio of k(cat) to K-m obtained from the saturation curve, is (1.0 +/- 0.2) x 10(6) M(-1) s(-1) at 3.0 +/- 0.2 degrees C. Results are discussed in the contest of the current state of knowledge of the mechanisms of the cyclooxygenase and peroxidase reactions of prostaglandin endoperoxide synthase.
