Inactivation of glutathione S-transferases by nitric oxide-derived oxidants: Exploring a role for tyrosine nitration

Reactive intermediates derived from nitric oxide ((NO)-N-.) 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 electrop hiles, are susceptible to inactivation by reactive nitrogen species (RNS). Treatment of isolated GSTs or rat liver homogenates with either peroxynitri te, the myeloper-oxidase/hydrogen peroxide/nitrite system, or tetranitromet hane, resulted in loss of GST activity with a concomitant increase in the f ormation of protein-associated 3-nitrotyrosine (NO(2)Tyr). This inactivatio n was only partially (< 25%) reversible by dithiothreitol, and exposure of GSTs to hydrogen peroxide or S-nitroso-glutathione was only partially inhib itory (< 25%) and did not result in protein nitration. Thus, irreversible m odifications such as tyrosine nitration may have contributed to GST inactiv ation by RNS. Since all GSTs contain a critical, highly conserved, active-s ite tyrosine residue, we postulated that this Tyr residue might present a p rimary target for nitration by RNS, thus leading to enzyme inactivation. To directly investigate this possibility, we analyzed purified mouse liver GS T-mu, following nitration by several RNS, by trypsin digestion, HPLC separa tion, and matrix-assisted laser desorption/ionization-time of flight analys is, to determine the degree of tyrosine nitration of individual Tyr residue s. Indeed, nitration was found to occur preferentially on several tyrosine residues located in and around the GST active site. However, RNS concentrat ions that resulted in near complete GST inactivation only caused up to 25% nitration of even preferentially targeted tyrosine residues. Hence, nitrati on of active-site tyrosine residues may contribute to GST inactivation by R NS, but is unlikely to fully account for enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidativ e) injury by environmental pollutants in association with inflammation. (C) 2001 Academic Press.