Diuretic drug binding to human glutathione transferase P1-1: potential role of Cys-101 revealed in the double mutant C47S/Y108V.
The diuretic drug ethacrynic acid (EA), both an inhibitor and substrate of pi class glutathione S-transferase (GST P1-1), has been tested in clinical trials as an adjuvant in chemotherapy. We recently studied the role of the active site residue Tyr-108 in binding EA to the enzyme and found that the analysis was complicated by covalent binding of this drug to the highly reactive Cys-47. Previous attempts to eliminate this binding by chemical modification yielded ambiguous results and therefore we decided here to produce a double mutant C47S/Y108V by site directed mutagenesis and further expression in Escherichia coli and the interaction of EA and its GSH conjugate (EASG) examined by calorimetric studies and X-ray diffraction. Surprisingly, in the absence of Cys-47, Cys-101 (located at the dimer interface) becomes a target for modification by EA, albeit at a lower conjugation rate than Cys-47. The Cys-47 → Ser mutation in the double mutant enzyme induces a positive cooperativity between the two subunits when ligands with affinity to G-site bind to enzyme. However, this mutation does not seem to affect the thermodynamic properties of ligand binding to the electrophilic binding site (H-site) and the thermal or chemical stability of this double mutant does not significantly affect the unfolding mechanism in either the absence or presence of ligand. Crystal structures of apo and an EASG complex are essentially identical with a few exceptions in the H-site and in the water network at the dimer interface.
Quesada-Soriano
Indalecio
I
Department of Physical Chemistry, Faculty of Experimental Sciences, University of Almería, La Cañada de San Urbano, 04120 Almería, Spain.
Parker
Lorien J
LJ
Primavera
Alessandra
A
Wielens
Jerome
J
Holien
Jessica K
JK
Casas-Solvas
Juan M
JM
Vargas-Berenguel
Antonio
A
Aguilera
Ana M
AM
Nuccetelli
Marzia
M
Mazzetti
Anna P
AP
Lo Bello
Mario
M
Parker
Michael W
MW
García-Fuentes
Luis
L
D019943:Amino Acid Substitution; D002151:Calorimetry; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics*; D004232:Diuretics / Q000378:metabolism*; D004789:Enzyme Activation; D004976:Ethacrynic Acid / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D051549:Glutathione S-Transferase pi / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D055503:Protein Multimerization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics
D004232:Diuretics; D050505:Mutant Proteins; D051549:Glutathione S-Transferase pi; D005978:Glutathione; D003545:Cysteine; D004976:Ethacrynic Acid