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

Abstract

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.

The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases

“…The present work shows that this residue is not uniformly important for all substrates : substitution of this tyrosine residue by a phenylalanine residue has a marked effect on the rate of conjugation of the good substrate CDNB, but very little effect on the rate of conjugation of the poor substrate ethacrynic acid. This could be explained if product dissociation were rate-limiting for ethacrynic acid conjugation, as shown very recently for wild-type rat GST A1-1 [36], but not for CDNB conjugation by human GST A1-1 ; the data would require that for ethacrynic acid product dissociation be rate-limiting both in the wild-type enzyme and in the Y9F mutant, where, judging by the results with CDNB, the chemical step appears to be at least 170-fold slower.…”

“…There is also evidence that the C-terminal residues (211-222) of the protein, which form a helix in the structure of the SbenzylGSH complex [8], are important for catalysis [20], and they have very recently been shown to determine the dissociation of the GSH-ethacrynic acid conjugate from rat GST A1-1 [36]. From the studies reported here of ligand binding and catalysis by the wild-type and ∆CT enzymes, we come to the following conclusions.…”

“…In terms of k cat \K m , CDNB is a better substrate than ethacrynic acid for the wild-type enzyme by a factor of 150, whereas in GST Y9F this ratio is decreased to 2 and in GST ∆CT to 0.001 -a striking reversal of specificity. A possible explanation for these different effects on CDNB and ethacrynic acid conjugation is suggested by the very recent report by Nieslanik et al [36] of the effects of C-terminal truncation of rat GST A1-1 on the conjugation of ethacrynic acid. They showed that product dissociation is rate-limiting for the wild-type rat enzyme, at least at room temperature and below, but not for a mutant in which residues 209-222 had been removed.…”

Ensemble Perspective for Catalytic Promiscuity

“…Construction of the rat GSTA1-1 (rGSTA1-1) ⌬209 -222 mutant has been described previously, as have the expression and purification (38,39). Activity of all purified enzymes was determined using the 1-chloro-2,4-dinitrobenzene (CDNB) assay (40).…”

Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of the Catalytic Activity of Glutathione Transferases

“…Similarly, in the human A1-1 GST (Alpha class), a rapid transition has been proposed involving motions of the flexible C-terminal segment that partially covers the G-site (31). Even the Mu class GST, where the active site is confined by two flexible loops, may display a similar fluctuating accessibility (32).…”