Implications of the ligandin binding site on the binding of non-substrate ligands to Schistosoma japonicum-glutathione transferase

The binding interactions between dimeric glutathione transferase from Schistosoma japonicum (Sj26GST) and bromosulfophthalein (BS) or 8-anilino-1-naphthalene sulfonate (ANS) were characterised by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Both ligands inhibit the enzymatic activity of Sj26GST in a non-competitive form. A stoichiometry of 1 molecule of ligand per mole of dimeric enzyme was obtained for the binding of these ligands. The affinity of BS is higher (K-d = 3.2 muM) than that for ANS (K-d = 195 muM). The thermodynamic parameters obtained by calorimetric titrations are pH-independent in the range of 5.5 to 7.5. The interaction process is enthalpically driven at all the studied temperatures. This enthalpic contribution is larger for the ANS anion than for BS. The strongly favourable enthalpic contribution for the binding of ANS to Sj26GST is compensated by a negative entropy change, due to enthalpy-entropy compensation. DeltaGdegrees remains almost invariant over the temperature range studied. The free energy change for the binding of BS to Sj26GST is also favoured by entropic contributions at temperatures below 32 degreesC, thus indicating a strong hydrophobic interaction. Heat capacity change obtained for BS (DeltaC(p)degrees = (-580.3 +/- 54.2) cal(.)K(-1) mol(-1)) is twofold larger (in absolute value) than for ANS (DeltaC(p)degrees = (-294.8 +/- 15.8) cal(.)K(-1) mol(-1)). Taking together the thermodynamic parameters obtained for these inhibitors, it can be argued that the possible hydrophobic interactions in the binding of these inhibitors to L-site must be accompanied by other interactions whose contribution is enthalpic. Therefore, the non-substrate binding site (designed as ligandin) on Sj26GST may not be fully hydrophobic. (C) 2004 Elsevier B.V. All rights reserved.