S-Nitrosation of Glutathione Transferase P1-1 Is Controlled by the Conformation of a Dynamic Active Site Helix

• Published in: The Journal of biological chemistry Vol. 288; no. 21; pp. 14973 - 14984
• Main Authors: Balchin, David, Wallace, Louise, Dirr, Heini W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.05.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Catalytic Domain; Covalent Regulation; Glutathione S-Transferase pi - chemistry; Glutathione S-Transferase pi - genetics; Glutathione S-Transferase pi - metabolism; Glutathione Transferase; Humans; Kinetics; Nitric Oxide; Nitrosation - physiology; Nitrosative Stress; Post-translational Modification; Protein Binding - physiology; Protein Dynamics; Protein Processing, Post-Translational - physiology; Protein Structure and Folding; Protein Structure, Secondary; S-Nitrosation; S-Nitrosoglutathione - chemistry; S-Nitrosoglutathione - metabolism; Transient Kinetics; Transient Kinetics; Protein Dynamics; Post-translational Modification; S-Nitrosation; Covalent Regulation; Nitrosative Stress; Nitric Oxide; Glutathione Transferase
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M113.462671

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. We used transient kinetic methods to determine a minimal mechanism for spontaneous S-nitrosoglutathione (GSNO)-mediated transnitrosation of human glutathione transferase (GST) P1-1, a major detoxification enzyme and key regulator of cell proliferation. Cys47 of GSTP1-1 is S-nitrosated in two steps, with the chemical step limited by a pre-equilibrium between the open and closed conformations of helix α2 at the active site. Cys101, in contrast, is S-nitrosated in a single step but is subject to negative cooperativity due to steric hindrance at the dimer interface. Despite the presence of a GSNO binding site at the active site of GSTP1-1, isothermal titration calorimetry as well as nitrosation experiments using S-nitrosocysteine demonstrate that GSNO binding does not precede S-nitrosation of GSTP1-1. Kinetics experiments using the cellular reductant glutathione show that Cys101-NO is substantially more resistant to denitrosation than Cys47-NO, suggesting a potential role for Cys101 in long term nitric oxide storage or transfer. These results constitute the first report of the molecular mechanism of spontaneous protein transnitrosation, providing insight into the post-translational control of GSTP1-1 as well as the process of protein transnitrosation in general. Background:S-Nitrosation is an emerging post-translational modification that is not yet well understood on a molecular level. Results: We propose a mechanism for S-nitrosation of glutathione transferase P1-1 by S-nitrosoglutathione. Conclusion: Cys101 is nitrosated in a single step, but Cys47 nitrosation is limited by the rate of helix 2 opening. Significance: Detailing the mechanism of spontaneous transnitrosation is crucial to understanding how protein S-nitrosation is controlled.