Stopped-Flow Kinetic Analysis of the Ligand-Induced Coil−Helix Transition in Glutathione S-Transferase A1-1:  Evidence for a Persistent Denatured State

• Published in: Biochemistry (Easton) 1999-05, Vol.38 (21), p.6971-6980
• Main Authors: Nieslanik, Brenda S, Dabrowski, Michael J, Lyon, Robert P, Atkins, William M
• Format: Article
• Language: English
• Subjects: Binding Sites - genetics; Glutathione Transferase - chemistry; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Humans; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - metabolism; Kinetics; Ligands; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Denaturation - genetics; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Spectrophotometry, Ultraviolet; Substrate Specificity - genetics; Viscosity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9829130

Structural studies have suggested that the glutathione S-transferase (GST) A1-1 isozyme contains a dynamic C-terminus which undergoes a ligand-dependent disorder−order transition and sequesters substrates within the active site. Here, the contribution of the C-terminus to the kinetics and thermodynamics of ligand binding and dissociation has been determined. Steady-state turnover rates of the wild type (WT) and a C-terminal truncated (Δ209−222) rGST A1-1 with ethacrynic acid (EA) were measured in the presence of variable concentrations of viscogen. The results indicate that a physical step involving segmental protein motion is at least partially rate limiting at temperatures between 10 and 40 °C for WT. Dissociation rates of the glutathione−ethacrynic acid product conjugate (GS−EA), determined by stopped-flow fluorescence, correspond to the steady-state turnover rates. In contrast, the chemical step governs the turnover reaction by Δ209−222, suggesting that the slow rate of product release for WT is controlled by the dynamics of the C-terminal coil−helix transition. In addition, the association reaction of WT rGST A1-1 with GS−EA established that the binding was biphasic and included ligand docking followed by slow isomerization of the enzyme−ligand complex. In contrast, binding of GS−EA to Δ209−222 was a monophasic, bimolecular reaction. These results indicate that the binding of GS−EA to WT rGST A1-1 proceeds via an induced fit mechanism, with a slow conformational step that corresponds to the coil−helix transition. However, the biphasic dissociation kinetics for the wild type, and the recovered kinetic parameters, suggest that a significant fraction of the [GST·GS−EA] complex (∼15%) retains a persistent disordered state at equilibrium.