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Arch Biochem Biophys. 1998 Jan 1;349(1):1-6.

Inactivation of glutathione peroxidase by peroxynitrite.

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  • 1Biodynamics Institute, Louisiana State University, Baton Rouge 70803-1800, USA.


Glutathione peroxidase (GSH-Px) is inactivated on exposure to peroxynitrite under physiologically relevant conditions. Stopped-flow kinetic studies show that the reaction between peroxynitrite and GSH-Px is first-order in each of the reactants, with an apparent second-order rate constant of 4.5 +/- 0.2 x 10(4) M-1 s-1 per monomer unit of enzyme. In good agreement with this value, GSH-Px inactivation experiments afford an apparent second-order rate constant of 1.8 +/- 0.1 x 10(4) M-1 s-1 per monomer unit of enzyme. The hydroxyl radical scavengers mannitol, DMSO, and benzoate (at 100 mM) afford only 8-12% protection of the enzyme, while addition of 25 mM bicarbonate results in 55% protection. The minimal protection by hydroxyl radical scavengers indicates, as expected, that hydroxyl radicals are not involved in the inactivation. Protection by bicarbonate occurs because peroxynitrite is rapidly trapped by CO2 to form the adduct nitrosoperoxycarbonate (ONOOCO2-), and/or other reactive species that preferentially decompose to nitrate rather than react with GSH-Px. The close agreement between the rate constants obtained from enzyme inactivation and from stopped-flow kinetics experiments suggests that the mechanism of the reaction between peroxynitrite and GSH-Px involves the oxidation of the ionized selenol of the selenocysteine residue in the enzyme's active site (E-Se-) by peroxynitrite. This reaction does not simply involve formation of the selenenic acid, E-SeOH, because E-SeOH is an intermediate in the catalytic cycle of the enzyme, and thus its formation cannot explain the inactivation we observe. Thus, the ionized selenol in the active site is transformed into a form of selenium that cannot easily be reduced back to the selenol.

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