The catalytic Tyr-9 of glutathione S-transferase A1-1 controls the dynamics of the C terminus

The glutathione S-transferase enzymes (GSTs) have a tyrosine or serine residue at their active site that hydrogen bonds to and stabilizes the thiolate anion of glutathione, GS(-). The importance of this hydrogen bond is obvious, in light of the enhanced nucleophilicity of GS(-)versus the protonated thiol. Several A-class GSTs contain a C-terminal segment that undergoes a ligand-dependent local folding reaction. Here, we demonstrate the effects of the Y9F substitution on binding affinity for glutathione conjugates and on rates of the order-disorder transition of the C terminus in rat GST A1-1. The equilibrium binding affinity of the glutathione conjugate, GS-NBD (NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole), was decreased from 4.09 mu M to 0.641 mu M upon substitution of Tyr-9 with Phe. This result was supported by isothermal titration calorimetry, with Kd values of 1.51 mu M and 0.391 mu M for wild type and Y9F, respectively. The increase in binding affinity for the mutant is associated with dramatic decreases in rates for the C-terminal order-disorder transition, based on a stopped-flow kinetic analysis. The same effects were observed, qualitatively, for a second GSH conjugate, GS-ethacrynic acid. Apparently, the phenolic hydroxyl group of Tyr-9 is critical for orchestrating C-terminal dynamics and efficient product release, in addition to its role in lowering the pK(a) of GSH.