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-depende nt disorder-order transition and sequesters substrates within the active si te. Here, the contribution of the C-terminus to the kinetics and thermodyna mics of ligand binding and dissociation has been determined. Steady-state t urnover rates of the wild type (WT) and a C-terminal truncated (Delta 209-2 22) rGST A1-1 with ethacrynic acid (EA) were measured in the presence of va riable concentrations of viscogen. The results indicate that a physical ste p 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 stopp ed-flow fluorescence, correspond to the steady-state turnover rates. In con trast, the chemical step governs the turnover reaction by Delta 209-222, su ggesting that the slow rate of product release for WT is controlled by the dynamics of the C-terminal coil-helix transition. In addition, the associat ion reaction of WT rGST A1-1 with GS-EA established that the binding was bi phasic and included ligand docking followed by slow isomerization of the en zyme-ligand complex. In contrast, binding of GS-EA to Delta 209-222 was a m onophasic, bimolecular reaction. These results indicate that the binding of GS-EA to WT rCST A1-1 proceeds via an induced fit mechanism, with a slow c onformational step that corresponds to the coil-helix transition. However, the biphasic dissociation kinetics for the wild type, and the recovered kin etic parameters, suggest that a significant fraction of the [GST.GS-EA] com plex (similar to 15%) retains a persistent disordered state at equilibrium.