Stopped-flow kinetic analysis of the ligand-induced coil-helix transition in glutathione S-transferase A1-1: evidence for a persistent denatured state.
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 (Delta209-222) 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 degrees 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 Delta209-222, 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 Delta209-222 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 ( approximately 15%) retains a persistent disordered state at equilibrium.
Nieslanik
B S
BS
Department of Medicinal Chemistry, University of Washington, Seattle 98195-7610, USA.
Dabrowski
M J
MJ
Lyon
R P
RP
Atkins
W M
WM
D001665:Binding Sites / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity / Q000235:genetics; D014783:Viscosity
D007527:Isoenzymes; D008024:Ligands; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha