A monomer form of the glutathione S-transferase Y7F mutant from Schistosoma japonicum at acidic pH |
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Authors: | Andújar-Sánchez Montserrat Clemente-Jimenez Josefa María Rodriguez-Vico Felipe Las Heras-Vazquez Francisco Javier Jara-Pérez Vicente Cámara-Artigas Ana |
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Affiliation: | Departamento Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, Spain. |
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Abstract: | 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 1.2x10(5)+/-6.4x10(3)M(-1) at pH 6.5 and 6.3x10(4)+/-1.25x10(3)M(-1) at pH 5). The stability of the SjGST-Y7F mutant was studied by urea induced unfolding techniques (DeltaG(W)=13.86+/-0.63kcalmol(-1) at pH 6.5 and DeltaG(W)=11.22+/-0.25kcalmol(-1) at pH 5) and the monomeric form characterized by means of size exclusion chromatography, fluorescence, and electrophoretic techniques. |
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Keywords: | Size exclusion chromatography Fluorescence Folding Isothermal titration calorimetry |
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