The Anomalous pK of Tyr-9 in Glutathione S-Transferase A1-1 Catalyzes Product Release

Abstract

The pK a of the catalytic Tyr-9 in glutathione S-transferase (GST) A1-1 is lowered from 10.3 to ϳ8.1 in the apoenzyme and ϳ9.0 with a GSH conjugate bound at the active site. However, a clear functional role for the unusual Tyr-9 pK a has not been elucidated. GSTA1-1 also includes a dynamic C terminus that undergoes a liganddependent disorder-to-order transition. Previous studies suggest a functional link between Tyr-9 ionization and C-terminal dynamics. Here we directly probe the role of Tyr-9 ionization in ligand binding and C-terminal conformation. An engineered mutant of rGSTA1-1, W21F/F222W, which contains a single Trp at the C terminus, was used as a fluorescent reporter of pH-dependent C-terminal dynamics. This mutant exhibited a pHdependent change in Trp-222 emission properties consistent with changes in C-terminal solvation or conformation. The apparent pK a values for the conformational transition were 7.9 ؎ 0.1 and 9.3 ؎ 0.1 for the apoenzyme and ligand-bound enzyme, respectively, in excellent agreement with the pK a for Tyr-9 in these states. The Y9F/W21F/F222W mutant, however, exhibited no such pH-dependent changes. Time-resolved fluorescence anisotropy studies revealed a ligand-dependent, Tyr-9-dependent, change in the order parameter of Trp-222. However, no pH dependence was observed. In equilibrium and pre-steady-state ligand binding studies, product conjugate had a decreased equilibrium binding affinity (K D ), concomitant with increased binding and dissociation rates, at higher pH values. Furthermore, the recovered pK a values for the pH-dependent microscopic rate constants ranged from 7.7 to 8.4, also in agreement with the pK a of Tyr-9. In contrast, the Y9F/ W21F/F222W mutant had no pH-dependent transition in K D or rate constants for ligand binding or dissociation. The combined results indicate that the macroscopic populations of "open" and "closed" states of the C terminus are not determined solely by the ionization state of Tyr-9. However, the rates of transition between these states are faster for the ionized Tyr-9. The ionized Tyr-9 states provide a parallel pathway for product dissociation, which is kinetically and thermodynamically favored. In silico kinetic models further support the functional role for the parallel dissociation pathway provided by ionized Tyr-9.

A Conserved N-capping Motif Contributes Significantly to the Stabilization and Dynamics of the C-terminal Region of Class Alpha Glutathione S-Transferases

“…It would appear that the reduction in catalytic efficiency is not due to an increase in K m CDNB but rather a decrease in k cat CDNB (not determined). A decrease in k cat CDNB , in turn, would not be due to a decreased rate of product release, because mobility-enhancing perturbations in the Cterminal region generally increase the rate of product release (12,51,53). Rather, it is most likely that the affinity of the H-site for CDNB is reduced, which is consistent with the link between productive binding of CDNB and the dynamics of the C-terminal region (9).…”

“…Therefore it is expected that the substitution of Asp-209 with a glycine would compromise the stability of helix 9. Tyr-9, the catalytic residue involved in the activation of GSH, displays an unusually low pK a of 8.1 (15) and is proposed to control the dynamics of the C-terminal region in GST A1-1, in that the region becomes more dynamic when Tyr-9 becomes ionized (50,51). This is unlikely a reason for the conformational change in the C-terminal region of D209G GST A1-1 at pH 6.5 because the pK a of Try-9 is not perturbed by the mutation (pK a is 8.2 for both wild-type and mutant; data not shown).…”

Tertiary Interactions Stabilise the C-terminal Region of Human Glutathione Transferase A1-1: a Crystallographic and Calorimetric Study

“…The identities of the groups that undergo protonation or deprotonation during inhibitor binding are not known. Although the pK a of the active-site Tyr9 in apo hGST A1-1 has been shown to increase from 8.1 to 9.0 during GTX binding, 28 this pK a effect is not significant at the pH of 6.5 used in our experimental studies. The I219A mutation does not perturb the pK a of Tyr9 in the apo form of the mutant protein.…”

“…25 The increased mobility of the C-terminal region and its positional differences between the wild-type and mutant apo proteins would also explain the reduction in functionality of I219A, given the tight coupling between catalytic function and the dynamics of the region. 10,11,14,16,17,24,25,27,28 Furthermore, desolvation of the active-site, important for maintaining the highly reactive thiolate form of activated glutathione, 18,38 is proposed to occur during the binding step of active-site ligands to hGST A1-1 and not during the ensuing ligand-induced localisation of the C-terminal region. 12 Crystallographic data, however, show that, while many water molecules are displaced from the active-site during ligand binding, desolvation of the site occurs during both steps.…”

“…14,22,23 Given that the catalytic and ligandin functions of GST A1-1 are tightly coupled to the C-terminal region, insight into the dynamics of the region is important to understanding the mechanisms by which the protein functions. 11,14,16,17,[24][25][26][27][28] In the present study, we investigated further the importance of tertiary interactions in determining the conformational dynamics of the C-terminal region in apo and ligand-complexed hGST A1-1.…”