Thermodynamic Description of the Effect of the Mutation Y49F on Human Glutathione Transferase P1-1 in Binding with Glutathione and the Inhibitor S-Hexylglutathione

Abstract

The thermodynamics of binding of both the substrate glutathione (GSH) and the competitive inhibitor S-hexylglutathione to the mutant Y49F of human glutathione S-transferase (hGST P1-1), a key residue at the dimer interface, has been investigated by isothermal titration calorimetry and fluorescence spectroscopy. Calorimetric measurements indicated that the binding of these ligands to both the Y49F mutant and wild-type enzyme is enthalpically favorable and entropically unfavorable over the temperature range studied. The affinity of these ligands for the Y49F mutant is lower than those for the wild-type enzyme due mainly to an entropy change. Therefore, the thermodynamic effect of this mutation is to decrease the entropy loss due to binding. Calorimetric titrations in several buffers with different ionization heat amounts indicate a release of protons when the mutant binds GSH, whereas protons are taken up in binding S-hexylglutathione at pH 6.5. This suggests that the thiol group of GSH releases protons to buffer media during binding and a group with low pK a (such as Asp 98 ) is responsible for the uptake of protons. The temperature dependence of the free energy of binding, ⌬G 0 , is weak because of the enthalpy-entropy compensation caused by a large heat capacity change. The heat capacity change is ؊199.5 ؎ 26.9 cal K ؊1 mol ؊1 for GSH binding and ؊333.6 ؎ 28.8 cal K ؊1 mol ؊1 for S-hexylglutathione binding. The thermodynamic parameters are consistent with the mutation Tyr 49 3 Phe, producing a slight conformational change in the active site.

Tertiary Interactions Stabilise the C-terminal Region of Human Glutathione Transferase A1-1: a Crystallographic and Calorimetric Study

“…The affinity for glutathione S-ethacrynic acid by wildtype protein is also an order of magnitude lower for the intermediate complex than for the final complex. 12 At equilibrium, the affinity of wildtype hGST A1-1 for GTX is also higher than that of wild-type hGST P1-1 (K d Z1.23 mM, 25 8C) 33 and of wild-type SjGST (K d Z1.6 mM, 25 8C). 39 The higher affinity of the former is due to the additional van der Waals contacts formed at the interface between its localised C-terminal region and the proteininhibitor complex.…”

“…This is consistent with NMR data indicating that the mobile C-terminal region in the apo wildtype protein samples alternative helix-like conformations, similar to those in ligand-complexed protein. 13 On the other hand, the large negative DH obs of complex formation between GTX and hGST P1-1 (K67.5 kJ/mol) 33 is a consequence of a ligand-induced folding of helix 2 at the activesite. 46 Since the binding of GTX to hGST P1-1 is not a rigid-body interaction, as previously suggested, 33 ligand-induced folding of helix 2 could contribute as much as K20 kJ/mol to the overall enthalpy term.…”

“…This is in agreement with the small to nearabsent proton-linkage effects reported for the binding of glutathione conjugates to a schistosomal GST 32 and hGST P1-1. 33 The effect of temperature on the binding of Shexylglutathione…”

Diuretic drug binding to human glutathione transferase P1‐1: potential role of Cys‐101 revealed in the double mutant C47S/Y108V

“…The experimental conditions and data analysis were similar to those described elsewhere (Té llez-Sanz et al, 2006;Quesada-Soriano et al, 2009). The binding of active site ligands to the C47S/Y108V GST P1-1 mutant quenches the intrinsic fluorescence of the enzyme as was described for wt GST P1-1 Ortiz-Salmeró n et al, 2003;Caccuri et al, 1991). Equilibrium unfolding/refolding experiments were performed as described (Aceto et al, 1992) at 258C in 20 mM sodium phosphate buffer, pH 7, containing 0.1 mM EDTA and 1 mM DTT.…”

Thermodynamics of Molecular Recognition by Calorimetry

“…This information is very valuable in drug design and cannot be obtained from structural or computational methods alone. In the first example we show the thermodynamics of binding of the substrate glutathione (GSH) and the competitive inhibitor S-hexylglutathione to the wt-enzyme and the Y49F mutant of the human glutathione S-transferase (hGST P1-1) (Ortiz-Salmerón et al, 2003). Structural studies revealed that two residues (Cys 47 and Tyr 49) located in a mobile helix, denoted as 2, could participate in intersubunit communication between active sites of the dimer.…”