FOLDING OF THE 4 DOMAINS AND DIMERIZATION ARE IMPAIRED BY THE GLY446-]GLU EXCHANGE IN HUMAN GLUTATHIONE-REDUCTASE - IMPLICATIONS FOR THE DESIGN OF ANTIPARASITIC DRUGS

Glutathione reductase (NADPH + GSSG + H+ --> NADP+ + 2GSH) is a homodi meric flavoenzyme of known geometry. Each subunit contains four well-d efined domains and contributes essential residues to the active sites; consequently, the monomer is expected to be inactive. As part of our program to develop dimerization inhibitors of human glutathione reduct ase (hGR) as antimalarial agents, we mutagenized the residues 446 and 447 which, together with their counterparts on the other subunit, repr esent the tightest contact between the subunits [Karplus, P. A., & Sch ulz, G. E. (1987) J. Mol. Biol. 195, 701-729]. Wild-type human glutath ione reductase and mutants of this protein were produced in plasmid-tr ansformed Escherichia coli SG5 cells. Active enzyme species, namely, w ild-type hGR, N-terminally truncated DELTA(1-15)hGR, and the point mut ant F447P-hGR, were purified by 2',5'-ADP-Sepharose chromatography and crystallization. Inactive mutants such as G446E-hGR or the double mut ants G446E/F447P-hGR and G446P/F447P-hGR were isolated by immunoadsorp tion chromatography. G446E/F447P-hGR was studied in detail. This mutan t behaved like a poorly folded monomeric protein, as indicated by the following properties: absence of the intersubunit disulfide bridge, Cy s90-Cys90'; failure to bind FAD; failure to bind NADPH and analogues t hereof, a short half-life (<4 min) in E. coli cells; and high suscepti bility to trypsin in vitro. The results suggest that the sequence arou nd G446 can control dimerization as well as domain folding. This is un expected since the FAD-binding domain and the NADPH-binding domain occ ur in many different enzymes and have been regarded as autonomous fold ing units. The role of dimer interface residues as a folding signal an d the prospect to develop inhibitors which render the target protein s usceptible to in vivo degradation are discussed.