A monomer form of the glutathione S-transferase Y7F mutant from Schistosoma japonicum at acidic pH.
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.
Andújar-Sánchez
Montserrat
M
Departamento Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, Spain.
Clemente-Jimenez
Josefa María
JM
Rodriguez-Vico
Felipe
F
Las Heras-Vazquez
Francisco Javier
FJ
Jara-Pérez
Vicente
V
Cámara-Artigas
Ana
A
D000818:Animals; D002151:Calorimetry; D002850:Chromatography, Gel; D019281:Dimerization; D004586:Electrophoresis; D004789:Enzyme Activation; D004795:Enzyme Stability; D005978:Glutathione / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D012549:Schistosoma japonicum / Q000737:chemistry* / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry*; D014508:Urea / Q000737:chemistry*
D007527:Isoenzymes; D014443:Tyrosine; D010649:Phenylalanine; D014508:Urea; D005978:Glutathione