Class-pi glutathione S-transferase is unable to regain its native conformation after oxidative inactivation by hydrogen peroxide.

The oxidation of protein sulphydryls to disulphides and their reduction back to sulphydryls is an early cellular response to oxidative stress. Hydrogen-peroxide-mediated oxidation of class-pi glutathione S-transferase results in the formation of disulphide bonds, which inhibits its catalytic function yet allows it to retain its non-substrate-ligand-binding properties. The overall hydrodynamic volume of the oxidised class-pi glutathione S-transferase type-1 homodimer (GSTP1-1) is decreased, and its tertiary-structural and secondary-structural elements are changed with respect to the native protein. Structural differences appear to be prominent in domain 1 of oxidised GSTP1-1, in that the exposure of both tryptophan residues is increased, while the electric potential about one of them is altered. Treatment of the oxidised protein with dithiothreitol or glutathione restores its enzymatic capabilities, albeit with lower specific activities for 1-chloro-2,4-dinitrobenzene and ethacrynic acid. The hydrophobic binding site (H-site) for electrophilic substrates is negatively affected in that the K(m) and catalytic-efficiency values are diminished significantly with respect to those values obtained for the native protein. The dithiothreitol-treated oxidised GSTP1-1 is able to regain its overall hydrodynamic volume; however, both its secondary-structural and tertiary-structural elements remain modified with respect to the native protein, as do both tryptophanyl environments. Furthermore, the oxidised glutathione S-transferase and dithiothreitol-treated oxidised glutathione S-transferase are less thermostable (tM = 55.5 degrees C and 56.3 degrees C, respectively) than the native enzyme (tM = 59 degrees C). These results indicate that the class-pi glutathione S-transferase is unable to regain its native conformation after oxidative inactivation.