A mechanistic study of self-inactivation of the peroxidase activity in prostaglandin H synthase-1.
Prostaglandin H synthase (PGHS) is a self-activating and self-inactivating enzyme. Both the peroxidase and cyclooxygenase activities have a limited number of catalytic turnovers. Sequential stopped-flow measurements were used to analyze the kinetics of PGHS-1 peroxidase self-inactivation during reaction with several different hydroperoxides. The inactivation followed single exponential kinetics, with a first-order rate constant of 0.2-0.5 s-1 at 24 degrees C. This rate was independent of the peroxide species and concentration used, strongly suggesting that the self-inactivation process originates after formation of Compound I and probably with Intermediate II, which contains an oxyferryl heme and a tyrosyl radical. Kinetic scan and rapid scan experiments were used to monitor the heme changes during the inactivation process. The results from both experiments converged to a simple, linear, two-step mechanism in which Intermediate II is first converted in a faster step (0.5-2 s-1) to a new compound, Intermediate III, which undergoes a subsequent slower (0.01-0.05 s-1) transition to a terminal species. Rapid-quench and high pressure liquid chromatography analysis indicated that Intermediate III likely retains an intact heme group that is not covalently linked with the PGHS-1 protein.
Wu
G
G
Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas 77030, USA.
Wei
C
C
Kulmacz
R J
RJ
Osawa
Y
Y
Tsai
A L
AL
D000818:Animals; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D051545:Cyclooxygenase 1; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D015289:Leukotrienes / Q000378:metabolism; D008054:Lipid Peroxides / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011462:Prostaglandins G / Q000378:metabolism; D012756:Sheep
D007527:Isoenzymes; D015289:Leukotrienes; D008054:Lipid Peroxides; D011462:Prostaglandins G; C038291:prostaglandin G2; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; D009195:Peroxidase; D051545:Cyclooxygenase 1; D011451:Prostaglandin-Endoperoxide Synthases