title: Detection of a higher oxidation state of manganese-prostaglandin endoperoxide synthase.
abstract: Addition of arachidonic acid or 5-phenyl-4-pentenylhydroperoxide to manganese-prostaglandin endoperoxide synthase (Mn-PGH synthase) produced a species with an absorbance maximum at 418 nm. This maximum is distinct from those of resting enzyme (372 and 468 nm) or reduced enzyme (434 nm). The formation of the 418 nm-absorbing species was observed immediately after the addition of hydroperoxide to enzyme but only after a 10-s lag period following addition of arachidonate. Mn-PGH synthase exhibited a peroxidase activity that was 0.8% that of Fe-PGH synthase. Addition of peroxidase reducing substrates to the oxidized form of Mn-PGH synthase diminished the absorbance at 418 nm. In the case of N,N,N',N'-tetramethylphenylenediamine, reduction of the 418 nm-absorbing species was accompanied by an increase in absorbance at 610 nm due to the oxidized form of the amine. Thus, the spectral and chemical properties of the 418 nm-absorbing species are consistent with its existence as a higher oxidation state of Mn-PGH synthase. Kinetic analysis indicated that formation of the higher oxidation state preceded or was coincident with oxygenation of the fatty acid substrate, eicosa-11,14-dienoic acid. The cyclooxygenase activity of Mn-PGH synthase was inhibited by the combination of glutathione and human plasma glutathione peroxidase at a glutathione peroxidase concentration 227-fold lower than the concentration that inhibited Fe-PGH synthase. The results suggest that Mn-PGH synthase forms a higher oxidation state following reaction with hydroperoxides added exogenously or generated endogenously from polyunsaturated fatty acid substrates. This higher oxidation state functions in the peroxidase catalytic cycle of Mn-PGH synthase, and its formation appears to be essential for activation of the cyclooxygenase catalytic cycle.
authors.affiliation: A. B. Hancock, Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
authors.forename: R
authors.initials: R
authors.lastname: Odenwaller
authors.forename: K R
authors.initials: KR
authors.lastname: Maddipati
authors.forename: L J
authors.initials: LJ
authors.lastname: Marnett
mesh_terms: D000475:Alkenes / Q000378:metabolism; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008345:Manganese / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism*; D010545:Peroxides / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep; D013053:Spectrophotometry
chemical_list: D000475:Alkenes; D001051:Apoenzymes; D010545:Peroxides; D016718:Arachidonic Acid; D008345:Manganese; C047254:5-phenylpent-4-enyl-1-hydroperoxide; D009195:Peroxidase; D011451:Prostaglandin-Endoperoxide Synthases; D005978:Glutathione