Thermodynamic analysis of the binding of glutathione and S-methyl glutathione to glutathione S-transferase over a range of temperatures

The glutathione transferases (EC 2.5.1.18) (GSTs) are a family of enzymes involved in the mechanism of cellular detoxification. They catalyze the nucleophilic attack of glutathione on the electrophilic centre of a number of toxic compounds. The cytosolic enzymes have two active sites per dimmer which behave independently of one another. The homodimeric (26 kDa per subunit) glutathione S-transferase from Schistosoma japonicum (Sj GST) is a member of one of the most important supergene families of enzymes involved in the phase II metabolism of electrophilic compounds. The glutathione-conjugates have greater solubility in water, facilitating their export from the cell, where they are metabolized via the mercapturate pathway and eventually excreted. It has been confirmed that each subunit in homodimeric SjGST contains two structural domains, an N-terminal and a C-terminal domain. The dimeric structure is required for the formation of two functional active sites (one per subunit). The binding properties of a glutathione S-transferase (EC 2.5.1.18) from Schistosoma japonicum to substrate glutathione (GSH) has been investigated by intrinsic fluorescence and isothermal titration calorimetry (ITC) at pH 6.5 over a temperature range of 15-30 C. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of GSH at pH 6.5. The effect of pH on the thermodynamics of GSH-GST interaction was studied. The binding isotherms were made at each pH in the presence of two different buffers and the number of protons at each pH was calculated. It is found that, the protons are taken up at pH 5 and 8.5, and are practically zero at pH 8, whereas they are released between pH 5.5 and 8. The behaviour shown at different pHs indicates that at least three groups must participate in the exchange of protons. Fluorimetric and calorimetric measurements indicate that GSH binds to two sites in the dimer of 26 kDa glutathione S-transferase from Schistosoma japonicum (Sj GST). On the other hand, noncooperativity for substrate binding to SjGST was detected over a temperature range of 15-30 C. Among thermodynamic parameters, whereas G remains practically invariant as a function of temperature, H and S are both decrease with an increase in temperature. While the binding is enthalpically favorable at all temperatures studied, at temperatures below 25 8C, Gis also favoured by entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attaining a value of zero at 24.3 C, and then becoming unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy-entropy compensation. The temperature dependence of the enthalpy change yields the heat capacity change (Cp  ) of  0.2380.04 kcal mol -1 K -1 . Moreover, the thermodynamic parameters, H, S, and G, for the competitive inhibitor Smethyl glutathione (SMG) have been investigated by ITC at pH 6.5 over a temperature range of 15-30 C. Calorimetric measurements in various buffer systems with different ionization enthalpies were performed and suggested that no protons were exchanged during the binding of SMG. The results revealed that this binding is both enthalpic and entropically favorable at all checked temperatures, resulting in an enthalpy-entropy compensation. The temperature dependence of the enthalpy change yields the heat capacity change of  0.1580.001 kcal moll -1