Reactions of prostaglandin endoperoxide synthase and its compound I with hydroperoxides

Abstract

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 104 M-1 s-1 using t-butyl hydroperoxide as the oxidant, (2.5 ± 0.1) x 106 M-1 s-1 for ethyl hydroperoxide and (5.1 ± 0.6) x 107 M-1 s-1 for m-chloroperoxybenzoic acid at pH 7.0, 6.7 ± 0.2 °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 G2 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 104 for t- butyl hydroperoxide, (3.5 ± 0.5) x 104 for hydrogen peroxide, (4.2 ± 0.2) x 104 for ethyl hydroperoxide, and (4.2 ± 0.3) x 105 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 G2 with compound I, calculated from the ratio of k(cat) to K(m) obtained from the saturation curve, is (1.0 ± 0.2) x 106 M-1 s-1 at 3.0 ± 0.2 °C. Results are discussed in the context of the current state of knowledge of the mechanisms of the cyclooxygenase and peroxidase reactions of prostaglandin endoperoxide synthase.