The C-terminus of glutathione S-transferase Al-1 is required for entropically-driven ligand binding

Binding of a hydrophobic glutathione product conjugate to rGST A1-1 proceed s via a two-step mechanism, including rapid ligand docking, followed by a s low isomerization to the final [GST.ligand] complex, which involves the loc alization of the flexible C-terminal helix. These kinetically resolved step s have been observed previously by stopped-flow fluorescence with the wild- type rGST Al-l, which contains a native Trp-21 approximately 20 Angstrom fr om the ligand binding site at the intrasubunit domain-domain interface. To confirm this binding mechanism, as well as elucidate the effects of truncat ion of the C-terminus, we have further characterized the binding and dissoc iation of the glutathione-ethacrynic acid product conjugate (GS-EA) to wild -type, F222W:W21F, and Delta 209-222 rGST A1-1 and wild-type hGST A1-1. Alt hough modest kinetic differences were observed between the hGST A1-1 and rG ST A1-1, stopped-flow binding studies with GS-EA verified that the two-step mechanism of ligand binding is not unique to the GST Al-l isoform from rat . An F222W:W21F rGST Al-l double mutant provides a direct fluorescence prob e of changes in the environment of the C-terminal residue. The observation of two relaxation times during ligand binding and dissociation to F222W:W21 F suggests that the C-terminus has an intermediate conformation following l igand docking, which is distinct from its conformation in the apoenzyme or localized helical state. For the wild-type, Delta 209-222, and F222W:W21F p roteins, variable-temperature stopped-flow experiments were performed and a ctivation parameters calculated for the individual steps of the binding rea ction. Activation parameters for the binding reaction coordinate illustrate that the C-terminus provides a significant entropic contribution to ligand binding, which is completely realized within the initial docking step of t he binding mechanism. In contrast, the slow isomerization step is enthalpic ally driven. The partitioning of entropic and enthalpic components of bindi ng energy was confirmed by isothermal titration calorimetry with wild-type and Delta 209-222 rGST A1-1.