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.
Highlights
Prostaglandin H synthase (PGHS)1 [1] catalyzes the first committed step in the biosynthesis of many important prostanoids
The results provide convincing evidence that the self-inactivation step occurs subsequent to formation of Intermediate II and is independent of peroxide structure and concentration
Kinetics of PGHS Peroxidase Inactivation during Reaction with EtOOH—PGHS was reacted with 250 M EtOOH for various times, and the surviving peroxidase activity was monitored by H2O2-dependent oxidation of guaiacol in a secondary reaction, as described under “Experimental Procedures” (Fig. 1A)
Summary
Guaiacol, and hemin were purchased from Sigma. Arachidonic acid was from Nu Chek Prep (Elysian, MN). Carryover of peroxide from the first stage reaction was evaluated by including EtOOH or 15-HPETE in the guaiacol/H2O2 solution used in the second stage reaction and found to have negligible effect on the observed peroxidase velocity (data not shown). Kinetics of PGHS Heme Spectral Changes Induced by Peroxide— Measurements were performed on the Bio-Sequential stopped-flow instrument in the kinetic scan-spectral reconstruction mode or on an Olis RSM-1000 rapid-scan stopped-flow instrument (On-Line Instrument Systems, Inc., Bogart, GA) The former approach was used to acquire data over shorter wavelength ranges (typically, 390 – 430 nm in 2-nm increments) and at lower enzyme concentrations. A set of published kinetic data and a mechanistic model for the PGHS peroxide reaction containing three spectral species [4] was used to validate the reliability of the SVD/global fitting software supplied with the Applied Photophysics and Olis instruments and achieved an excellent agreement. The rate of reduction of tyrosine radical in species Fe(IV)Tyr1⁄7 was assumed to be the same as that in Fe(III)Tyr1⁄7
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