REACTIONS OF PROSTAGLANDIN ENDOPEROXIDE SYNTHASE AND ITS COMPOUND-I WITH HYDROPEROXIDES

The reactions of native prostaglandin endoperoxide synthase with struc turally different hydroperoxides have been investigated by using kinet ic spectrophotometric scan and conventional and sequential mixing stop ped-flow experiments. The second order rate constants for compound I f ormation are (5.9 +/- 0.1) x 10(4) M(-1) s(-1) using t-butyl hydropero xide as the oxidant, (2.5 +/- 0.1) x 10(6) M(-1) s(-1) for ethyl hydro peroxide and (5.1 +/- 0.6) x 10(7) M(-1) s(-1) for m-chloroperoxybenzo ic acid at pH 7.0, 6.7 +/- 0.2 degrees C, and ionic strength 0.1 M. Se quential mixing, transient state experiments show for the first time t hat all hydroperoxides reduce compound I in a bimolecular reaction. Et hyl hydroperoxide, t-butyl hydroperoxide, and m-chloroperoxybenzoic ac id react directly with compound I. The natural substrate prostaglandin G(2) forms a transient complex with compound I before the reduction s tep occurs. Therefore, compound I initially transforms to compound II, not to the compound I-tyrosyl radical. Second order rate constants fo r the reactions of compound I are (2.9 +/- 0.2) x 10(4) for t-butyl hy droperoxide, (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-chl oroperoxybenzoic 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 obtaine d 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 curr ent state of knowledge of the mechanisms of the cyclooxygenase and per oxidase reactions of prostaglandin endoperoxide synthase.