Peroxynitrite modification of glutathione reductase: modeling studies and kinetic evidence suggest the modification of tyrosines at the glutathione disulfide binding site (original) (raw)

1996, "Protein Engineering, Design and Selection"

The catalytic properties of glutathione reductase for its substrate, glutathione disulfide, were altered following a 60 s exposure to a 100-fold molar excess of peroxynitrite; the X M value was increased by ~2.5-fold and the V ma% value was decreased by ~1.7-fold. The kinetic alterations are thought to result from nitrotyrosine formation as the intrinsic Tyr fluorescence is diminished. The UV-visible spectrum of glutathione reductase exhibited absorbance at -423 nm, characteristic of nitrotyrosine. In addition, the presence of nitrotyrosine has been detected by Western immunoblots with an anti-nitrotyrosine antibody. The peroxynitrite-induced inactivation is not observed in the presence of excess glutathione disulfide. However, excess NADPH offered no protection against peroxynitriteinduced inactivation. These observations suggest that the modification of -1.8 Tyr per subunit, at or near the glutathione disulfide binding domain, probably results in the observed catalytic alterations. To test this hypothesis, the two tyrosines closest to the glutathione disulfide binding domain (Tyrll4 and TyrlO6), as indicated by the X-ray crystallographic data [Karplus and Schulz (1989) J. Biol Chenu, 210,163-180], were each converted to nitrotyrosines by molecular modeling and the structure energy was minimized. These theoretical calculations indicate that the bond lengths between Tyrll4-O and the Gly-N and Cys II-N of glutathione disulfide bound to glutathione reductase (Karplus and Schulz, 1989) increased by 3.0 and 4.3 A, respectively, upon nitration. In the case of TyrlO6, the O-Cys II-N distance also increases by -1.6 A. The loss of these hydrogen bonding contacts is likely to result in the observed catalytic alterations upon reaction with peroxynitrite. Keywords: glutathione reductase/nitrotyrosine/peroxynitrite