Interaction of acetaminophen with myeloperoxidase intermediates: optimum stimulation of enzyme activity.
The myeloperoxidase-H2O2 system constitutes an effective physiological defense mechanism because of its ability to oxidize Cl- to HOCl, a powerful oxidizing agent, via the enzyme intermediate compound I. Two other oxidized intermediates, compounds II and III, are inactive in generation of HOCl. Acetaminophen, a safe drug at therapeutic doses but toxic at higher doses, was found to react with the oxidized intermediates of myeloperoxidase. Using steady-state kinetics on the chlorination of monochlorodimedon to measure the activity of myeloperoxidase, our study reveals that acetaminophen stimulates the chlorinating activity of the enzyme and optimum stimulation is achieved at about 30 microM. Increasing the concentration further causes a decline in the chlorination rate. The increase in enzyme activity at lower acetaminophen concentrations is accounted for by the increased turnover of compounds II and III to native enzyme, while the decline at higher acetaminophen concentrations is explained by the competition of acetaminophen with Cl- for compound I. Rapid scan and transient state kinetic results on the reaction of compound II and acetaminophen show that: (i) compound II does not pass through any other intermediate when acetaminophen reduces it back to native enzyme; and (ii) a simple binding interaction before enzyme reduction is involved. An apparent dissociation constant of 1.3 +/- 0.3 x 10(-4) M and a first-order rate constant for reduction of 37 +/- 4 s-1 were determined at 25 degrees C. Conventional spectral scans of the reaction between compound III and acetaminophen indicate that compound III goes back to native enzyme without any detectable intermediate. The rate of this reaction levels off at higher acetaminophen concentration. Rapid scans reveal that the reduction of compound I to compound II is faster in the presence of acetaminophen. Since the therapeutic concentrations of acetaminophen in man range approximately from 50 to 150 microM, the results of this study indicate that stimulation of myeloperoxidase activity is achieved within the safe dosage of the drug.
Department of Chemistry, University of Alberta, Edmonton, Canada.
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Marquez
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Dunford
D000082:Acetaminophen / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002713:Chlorine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005609:Free Radicals; D066298:In Vitro Techniques; D007700:Kinetics; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism*; D013057:Spectrum Analysis
D005609:Free Radicals; D000082:Acetaminophen; D002713:Chlorine; D009195:Peroxidase