Reactive intermediates derived from
nitric oxide ((*)NO) are thought to play a contributing role in disease states associated with
inflammation and
infection. We show here that
glutathione S-
transferases (
GSTs), principal
enzymes responsible for detoxification of endogenous and exogenous electrophiles, are susceptible to inactivation by
reactive nitrogen species (RNS). Treatment of isolated
GSTs or rat liver homogenates with either
peroxynitrite, the
myeloperoxidase/
hydrogen peroxide/
nitrite system, or
tetranitromethane, resulted in loss of GST activity with a concomitant increase in the formation of
protein-associated
3-nitrotyrosine (
NO(2)Tyr). This inactivation was only partially (<25%) reversible by
dithiothreitol, and exposure of
GSTs to
hydrogen peroxide or
S-nitrosoglutathione was only partially inhibitory (<25%) and did not result in
protein nitration. Thus, irreversible modifications such as
tyrosine nitration may have contributed to GST inactivation by RNS. Since all
GSTs contain a critical, highly conserved, active-site
tyrosine residue, we postulated that this Tyr residue might present a primary target for nitration by RNS, thus leading to
enzyme inactivation. To directly investigate this possibility, we analyzed purified mouse liver GST-mu, following nitration by several RNS, by
trypsin digestion, HPLC separation, and matrix-assisted
laser desorption/ionization-time of flight analysis, to determine the degree of
tyrosine nitration of individual Tyr residues. Indeed, nitration was found to occur preferentially on several
tyrosine residues located in and around the GST active site. However, RNS concentrations that resulted in near complete GST inactivation only caused up to 25% nitration of even preferentially targeted
tyrosine residues. Hence, nitration of active-site
tyrosine residues may contribute to GST inactivation by RNS, but is unlikely to fully account for
enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidative) injury by
environmental pollutants in association with
inflammation.