A monomer form of the glutathione S-transferase Y7F mutant from Schistosoma japonicum at acidic pH
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Andújar Sánchez, Montserrat; Clemente Jiménez, Josefa María; Rodríguez Vico, Felipe; Las Heras Vázquez, Francisco Javier; Jara Pérez, Vicente; [et al.]Fecha
2004Resumen
Dissociation and unfolding of homodimeric glutathione S-transferase Y7F mutant from Schistosoma japonicum (SjGST-Y7F)
were investigated at equilibrium using urea as denaturant. The conserved residue Tyr7 plays a central role in the catalytic mechanism
and the mutation Tyr–Phe yields an inactive enzyme that is able to bind the substrate GSH with a higher binding constant than the
wild type enzyme. Mutant SjGST-Y7F is a dimer at pH 6 or higher and a stable monomer at pH 5 that binds GSH (K value of
1.2 105 6.4 103M 1 at pH 6.5 and 6.3 104 1.25 103M 1 at pH 5). The stability of the SjGST-Y7F mutant was studied by
urea induced unfolding techniques (DGW ¼ 13:86 0:63 kcal mol 1 at pH 6.5 and DGW ¼ 11:22 0:25 kcal mol 1 at pH 5) and the
monomeric form characterized by means of size exclusion chromatography, fluorescence, and electrophoretic techniques.
2003 Elsevier Inc. All rights reserved.
Palabra/s clave
Size exclusion chromatography
Fluorescence
Folding
Isothermal titration calorimetry