CHARACTERIZATION OF A NONCOMPETITIVE INHIBITOR OF HUMAN GLUTATHIONE REDUCTASE. Savvas N. Savvides and P.Andrew Karplus, Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA

We have determined the crystal structure of a complex between human glutathione reductase (hGR) and its noncompetitive inhibitor 6-hydroxy-3oxo-3H-xanthene-9-propionic acid (XAN) [Kis=27 mM, Kii=48 mM with respect to oxidized glutathione (GSSG), and Kis=144 mM, Kii=176 mM with respect to NADPH]. The structure has been refined to an R-factor of 0.158 at 2.0 Å resolution, and reveals XAN bound in the large cavity present at the hGR dimer interface where it does not overlap the glutathione binding site. The inhibitor causes extensive structural changes at its binding site that primarily involve amino acid residues from a thirty residue a-helix which lines the cavity and contributes to the active site of hGR. Despite the lack of physical overlap of XAN with the GSSG binding site, no GSSG binding is seen in soaks carried out with high XAN and GSSG concentrations, suggesting that some subtle interaction between the sites exists. An earlier crystallographic analysis on the complex between hGR and 3,7-diamino-2,8-dimethyl-5-phenyl-phenazinium chloride (safranin) showed that safranin bound at this same site. We have found that safranin also inhibits hGR in a noncompetitive fashion but it binds about 16 times less tightly (Kis=453 mM, Kii=586 mM with respect to GSSG) than XAN, and does not preclude the binding of GSSG in the crystal. Despite our relatively detailed crystallographic results pertaining to the interactions XAN makes with the enzyme, the inhibition mechanism remains unclear. Although in structure-based drug design competitive inhibitors are usually targetted, XAN's binding to a well defined site in hGR that is not well conserved among homologs of hGR suggests that noncompetitive inhibitors could also serve as lead compounds for structure-based drug design, in particular as components of chimeric inhibitors.