The role of the hydroxyl group of tyrosine 6 in the binding of Schistosoma japonicum glutathione S-transferase has been investigated by isothermal titration calorimetry (ITC). A site-specific replacement of this residue with phenylalanine produces the Y6F mutant, which shows negative cooperativity for the binding of reduced glutathione (GSH). Calorimetric measurements indicated that the binding of GSH to Y6F dimer is enthalpically driven over the temperature range investigated. A concomitant net uptake of protons upon binding of GSH to Y6F mutant was detected carrying out calorimetric experiments in various buffer systems with different heats of ionization. The entropy change is favorable at temperatures below 26°C for the first site, being entropically favorable at all temperatures studied for the second site. The enthalpy change of binding is strongly temperature-dependent, arising from a large negative ΔC°p1=-3.45±0.62kJK-1mol-1for the first site, whereas a small ΔC°p2=-0.33±0.05kJK-1mol-1for the second site was obtained. This large heat capacity change is indicative of conformational changes during the binding of substrate. © 2003 Elsevier B.V. All rights reserved.
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