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A mechanistic study of self-inactivation of the peroxidase activity in prostaglandin H synthase-1.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 1999 Apr 02; Vol. 274 (14), pp. 9231-7. - Publication Year :
- 1999
-
Abstract
- 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.
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 274
- Issue :
- 14
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 10092596
- Full Text :
- https://doi.org/10.1074/jbc.274.14.9231