Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding

In addition to their catalytic functions, cytosolic glutathioneS-transferases (GSTs) are a major reserve of high-capacity binding proteins for a large variety of physiological and exogenous non-substrate compounds. This ligandin function has implicated GSTs in numerous ligand-uptake, -transport and -storage processes. The binding of non-substrate ligands to GSTs can inhibit catalysis. In the present study, the energetics of the binding of the non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to wild-type human class Alpha GST with two type-1 subunits (hGSTA1-1) and its ΔPhe-222 deletion mutant were studied by isothermal titration calorimetry. The stoichiometry of binding to both proteins is one ANS molecule per GST subunit with a greater affinity for the wild-type (K_d=65μM) than for the ΔPhe-222 mutant (K_d=105μM). ANS binding to the wild-type protein is enthalpically driven and it is characterized by a large negative heat-capacity change, ΔC_p. The negative ΔC_p value for ANS binding indicates a specific interface with a significant hydrophobic component in the protein—ligand complex. The negatively charged sulphonate group of the anionic ligand is apparently not a major determinant of its binding. Phe-222 contributes to the binding affinity for ANS and the hydrophobicity of the binding site.