title: Energetics of ligand binding to human glutathione transferase A1-1: Tyr-9 associated localisation of the C-terminal helix is ligand-dependent.
abstract: A C-terminal helix (α9) adjacent to the active site on each subunit is a structural feature unique to the alpha isoform of glutathione transferases which contributes to the catalytic and ligandin functions of the enzyme. The ionisation state of Tyr-9, a residue critical to catalysis, influences α9 dynamics, although the mechanism is poorly understood. In this study, isothermal titration calorimetry was used to probe the binding energetics of G-site (glutathione and glutathione sulfonate) and H-site (ethacrynic acid) ligands to wild-type and a Y9F mutant of human glutathione transferase A1-1. Although previous studies have reported a favourable entropic component to the binding of conjugates occupying both sites, our data reveal that ligand binding is enthalpically driven when either the G- or H-site is occupied independently. Also, heat capacity changes demonstrate that α9 is fully localised by H-site but not G-site occupation. The Tyr-9 hydroxyl group contributes significantly to ligand binding energetics, although the effect differs between the two binding sites. G-site binding is made slightly enthalpically more favourable and entropically less favourable by the Y9F mutation. Binding to the H-site is more dramatically affected, with the K(d) for ethacrynic acid increasing 5 fold despite a more favourable ΔS. The heat capacity change is more negative for G-site binding in the absence of the Tyr-9 hydroxyl (ΔΔC(p)=-0.73 kJ mol(-1) K(-1)), but less negative for H-site binding to the Y9F mutant (ΔΔC(p)=0.63 kJ mol(-1) K(-1)). This suggests that the relationship between Tyr-9 and α9 is not independent of the ligand. Rather, Tyr-9 appears to function in orienting the ligand optimally for α9 closure.
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, South Africa.
authors.forename: David
authors.initials: D
authors.lastname: Balchin
authors.forename: Heini W
authors.initials: HW
authors.lastname: Dirr
authors.forename: Yasien
authors.initials: Y
authors.lastname: Sayed
mesh_terms: D019943:Amino Acid Substitution; D002151:Calorimetry; D020134:Catalytic Domain; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D007527:Isoenzymes; D008024:Ligands; D014443:Tyrosine; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha