Role of mutation Y6F on the binding properties of Schistosoma japonicum glutathione S-transferase

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 f...

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Bibliographic Details
Published in:International journal of biological macromolecules 2003-09, Vol.32 (3), p.67-75
Main Authors: Yassin, Zeyad, Ortiz-Salmerón, Emilia, Clemente-Jiménez, M.José, Barón, Carmen, Garcı́a-Fuentes, Luis
Format: Article
Language:English
Subjects:
Amino Acid Substitution - genetics
Animals
Binding
Binding Sites
Calorimetry
Fluorescence
Glutathione
Glutathione S-transferase
Glutathione Transferase - chemistry
Glutathione Transferase - genetics
Glutathione Transferase - metabolism
Kinetics
Microcalorimetry
Phenylalanine - genetics
Protein Binding
Protein Folding
S-Methylglutathione
Schistosoma japonicum
Schistosoma japonicum - enzymology
Temperature
Thermodynamics
Tyrosine - genetics
Unfolding
Urea - pharmacology
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Summary: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 Δ C° p1=−3.45±0.62 kJ K −1 mol −1 for the first site, whereas a small Δ C° p2=−0.33±0.05 kJ K −1 mol −1 for the second site was obtained. This large heat capacity change is indicative of conformational changes during the binding of substrate.
ISSN:0141-8130
1879-0003
DOI:10.1016/S0141-8130(03)00039-4