title: 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 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 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 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 G2 with compound I, calculated from the ratio of kcat to Km 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 context of the current state of knowledge of the mechanisms of the cyclooxygenase and peroxidase reactions of prostaglandin endoperoxide synthase.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Bakovic
authors.forename: H B
authors.initials: HB
authors.lastname: Dunford
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001565:Benzoates / Q000378:metabolism; D003373:Coumaric Acids / Q000378:metabolism; D016861:Cyclooxygenase Inhibitors / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism; D007700:Kinetics; D008297:Male; D010545:Peroxides / Q000378:metabolism*; D011462:Prostaglandins G / Q000378:metabolism; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep; D014443:Tyrosine / Q000737:chemistry
chemical_list: D001565:Benzoates; D003373:Coumaric Acids; D016861:Cyclooxygenase Inhibitors; D010545:Peroxides; D011462:Prostaglandins G; D016718:Arachidonic Acid; D014443:Tyrosine; C038291:prostaglandin G2; C004999:ferulic acid; C017611:peroxybenzoic acid; D006735:Horseradish Peroxidase