Abstract
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 microm to 0.641 microm upon substitution of Tyr-9 with Phe. This result was supported by isothermal titration calorimetry, with K(d) values of 1.51 microm and 0.391 microm 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.
Highlights
The A-class glutathione S-transferase enzymes (GSTs) are unique in that several isoforms contain a dynamic C terminus that undergoes a ligand-dependent disorder-to-order transition [25, 26]
We hypothesized on the basis of high pressure experiments that the Tyr-9 hydrogen bond controls the dynamics of the C terminus [28]
In accordance with previous results with the glutathioneethacrynic acid product conjugate (GS-EA), we propose that the initial step, k1, corresponds to the docking of GS-NBD within the active site to yield [GSTzGS-NBD], a complex that has a disordered C terminus
Summary
Chemicals and Instrumentation—GSH, NBD-Cl, and MES were obtained from Sigma. Spectro/Por dialysis membrane was obtained from Spectrum Medical Industries, Inc. (Houston, TX). 1 mM GST (300 ml) was dialyzed against an equal volume of 0.5–50 mM GS-NBD in a 25 °C horizontal shaker water bath. GST solutions (100 –200 mM) were titrated with 10-ml injections of GS-NBD (1- 2 mM), and the heat flow between the reaction vessel and an isothermal heat sink was monitored. Binding rates were measured by the decrease in protein fluorescence after mixing an equal volume of 2 mM GST and 20 – 400 mM GS-NBD. Dissociation of product from the [GSTzGS-NBD] complex was followed by the increase in protein fluorescence after rapidly mixing 2 mM complex with an equal volume of 5 mM glutathione sulfonic acid (GSO32) at 15 °C. Dissociation rate constants were obtained directly from a fit to the single exponential equation (Equation 1)
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