Crystallographic and thermodynamic analysis of the binding of S-octylglutathione to the Tyr 7 to Phe mutant of glutathione S-transferase from Schistosoma japonicum.
Glutathione S-transferases are a family of multifunctional enzymes involved in the metabolism of drugs and xenobiotics. Two tyrosine residues, Tyr 7 and Tyr 111, in the active site of the enzyme play an important role in the binding and catalysis of substrate ligands. The crystal structures of Schistosoma japonicum glutathione S-transferase tyrosine 7 to phenylalanine mutant [SjGST(Y7F)] in complex with the substrate glutathione (GSH) and the competitive inhibitor S-octylglutathione (S-octyl-GSH) have been obtained. These new structural data combined with fluorescence spectroscopy and thermodynamic data, obtained by means of isothermal titration calorimetry, allow for detailed characterization of the ligand-binding process. The binding of S-octyl-GSH to SjGST(Y7F) is enthalpically and entropically driven at temperatures below 30 degrees C. The stoichiometry of the binding is one molecule of S-octyl-GSH per mutant dimer, whereas shorter alkyl derivatives bind with a stoichiometry of two molecules per mutant dimer. The SjGST(Y7F).GSH structure showed no major structural differences compared to the wild-type enzyme. In contrast, the structure of SjGST(Y7F).S-octyl-GSH showed asymmetric binding of S-octyl-GSH. This lack of symmetry is reflected in the lower symmetry space group of the SjGST(Y7F).S-octyl-GSH crystals (P6(3)) compared to that of the SjGST(Y7F).GSH crystals (P6(3)22). Moreover, the binding of S-octyl-GSH to the A subunit is accompanied by conformational changes that may be responsible for the lack of binding to the B subunit.
Andújar-Sánchez
Montserrat
M
Departamento Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, España.
Smith
Alex W
AW
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; D001667:Binding, Competitive / Q000235:genetics; D002151:Calorimetry; D003460:Crystallization; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D012549:Schistosoma japonicum / Q000201:enzymology* / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics; D014443:Tyrosine / Q000235:genetics
D004791:Enzyme Inhibitors; D021122:Protein Subunits; C059196:S-octylglutathione; D014443:Tyrosine; D010649:Phenylalanine; D005982:Glutathione Transferase; D005978:Glutathione; C033550:hexylglutathione; C017514:S-methyl glutathione