INACTIVATION OF GLUTATHIONE-PEROXIDASE BY PEROXYNITRITE

Glutathione peroxidase (GSH-Px) is inactivated on exposure to peroxyni trite under physiologically relevant conditions, Stopped-flow kinetic studies show that the reaction between peroxynitrite and GSH-Px is fir st-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 aff ord 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 mann itol, DMSO, and benzoate (at 100 mM) afford only 8-12% protection of t he enzyme, while addition of 25 mM bicarbonate results in 55% protecti on, The minimal protection by hydroxyl radical scavengers indicates, a s expected, that hydroxyl radicals are not involved in the inactivatio n. Protection by bicarbonate occurs because peroxynitrite is rapidly t rapped by CO2 to form the adduct nitrosoperoxycarbonate (ONOOCO2), and /or other reactive species that preferentially decompose to nitrate ra ther than react with GSH-Px. The close agreement between the rate cons tants obtained from enzyme inactivation and from stopped-flow kinetics experiments suggests that the mechanism of the reaction between perox ynitrite and GSH-Px involves the oxidation of the ionized selenol of t he selenocysteine residue in the enzyme's active site (E-Se-) by perox ynitrite. This reaction does not simply involve formation of the selen enic acid, E-SeOH, because E-SeOH is an intermediate in the catalytic cycle of the enzyme, and thus its formation cannot explain the inactiv ation we observe, Thus, the ionized selenol in the active site is tran sformed into a form of selenium that cannot easily be reduced back to the selenol. (C) 1998 Academic Press.