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 Soriano0, I., Parker, L., Primavera, A., Wielens, J., Holien, J., Casas Solvas, J., et al. (2011). Diuretic drug binding to human glutathione transferase P1-1: potential role of Cys-101 revealed in the double mutant C47S/Y108V. JOURNAL OF MOLECULAR RECOGNITION, 24(2), 220-234 [10.1002/jmr.1040].

Diuretic drug binding to human glutathione transferase P1-1: potential role of Cys-101 revealed in the double mutant C47S/Y108V

MAZZETTI, ANNA PAOLA;LO BELLO, MARIO;
2011-01-01

Abstract

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.
2011
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore BIO/12 - BIOCHIMICA CLINICA E BIOLOGIA MOLECOLARE CLINICA
English
Con Impact Factor ISI
Mutant Proteins; Ethacrynic Acid; Thermodynamics; Enzyme Activation; Glutathione; Models, Molecular; Humans; Hydrogen-Ion Concentration; Diuretics; Protein Multimerization; Structure-Activity Relationship; Glutathione S-Transferase pi; Cysteine; Kinetics; Calorimetry; Crystallography, X-Ray; Substrate Specificity; Mutation; Amino Acid Substitution
Quesada Soriano0, I., Parker, L., Primavera, A., Wielens, J., Holien, J., Casas Solvas, J., et al. (2011). Diuretic drug binding to human glutathione transferase P1-1: potential role of Cys-101 revealed in the double mutant C47S/Y108V. JOURNAL OF MOLECULAR RECOGNITION, 24(2), 220-234 [10.1002/jmr.1040].
Quesada Soriano0, I; Parker, L; Primavera, A; Wielens, J; Holien, J; Casas Solvas, J; Vargas Berenguel, A; Aguilera, A; Nuccetelli, M; Mazzetti, Ap; L...espandi
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/55259
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