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

• DOI: 10.1002/jmr.1040
• Publication Date: 2010-06-10T08:30:42Z
• Authors: Indalecio Quesada...; Lorien J. Parker; Alessandra Primavera; Jerome Wielens; Jessica K. Holien; Juan M. Casas‐Solvas; Antonio Vargas‐Be...; Ana M. Aguilera; Marzia Nuccetelli; Anna P. Mazzetti; Mario Lo Bello; Michael W. Parker; Luis García‐Fuentes
• Keywords: Glutathione S-transferase; Transferase

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 adjuvant chemotherapy. We recently studied the role active site residue Tyr-108 binding EA to enzyme found that analysis was complicated by covalent this highly reactive Cys-47. Previous attempts eliminate chemical modification yielded ambiguous results therefore we decided here produce a double mutant C47S/Y108V directed mutagenesis further expression Escherichia coli interaction its GSH conjugate (EASG) examined calorimetric studies X-ray diffraction. Surprisingly, absence Cys-47, Cys-101 (located at dimer interface) becomes target for EA, albeit lower conjugation rate than Cys-47 → Ser mutation induces positive cooperativity between two subunits when ligands with affinity G-site bind enzyme. However, does not seem affect thermodynamic properties ligand electrophilic (H-site) thermal or stability significantly unfolding mechanism either presence ligand. Crystal structures apo EASG complex are essentially identical few exceptions H-site water network interface.