Regulation of glutathione transferase P1-1 by S-nitrosation
S-Nitrosation is a post-translational modification of protein cysteine residues, which occurs in response to cellular oxidative stress. Although it is increasingly being linked to physiologically important processes, the molecular basis for protein regulation by this modification remains poorly understood. Biophysical methods were used to elucidate the mechanism and molecular consequences of S-nitrosation of glutathione transferase (GST) P1-1, a ubiquitous homodimeric detoxification enzyme and important target for cancer therapeutics. Transient kinetic techniques, isothermal titration calorimetry and protein engineering were used to develop a minimal mechanism for S-nitrosation of GSTP1-1, the first for any protein. Cys47 of GSTP1-1 is S-nitrosated according to a conformational selection mechanism, with the chemical step limited by a pre-equilibrium between the open and closed conformations of a dynamic helix at the active site. Cys101, in contrast, is Snitrosated in a single step but is subject to negative cooperativity due to steric hindrance at the dimer interface. S-Nitrosation at Cys47 and Cys101 was found to reduce the detoxification activity of GSTP1-1 by 94%. Circular dichroism spectroscopy, acrylamide quenching and amide hydrogen-deuterium exchange mass spectrometry experiments revealed that the loss of activity is due to the introduction of local disorder at the active site. Furthermore, the modification destabilises domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation, and provide general insight into the mechanism of S-nitrosation and its effect protein stability and dynamics.