Folding of the four 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 homodimeric flavoenzyme of known geometry. Each subunit contains four well-defined 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 reductase (hGR) as antimalarial agents, we mutagenized the residues 446 and 447 which, together with their counterparts on the other subunit, represent the tightest contact between the subunits [Karplus, P. A., & Schulz, G. E. (1987) J. Mol. Biol. 195, 701-729]. Wild-type human glutathione reductase and mutants of this protein were produced in plasmid-transformed Escherichia coli SG5 cells. Active enzyme species, namely, wild-type hGR, N-terminally truncated delta(1-15)hGR, and the point mutant F447P-hGR, were purified by 2',5'-ADP-Sepharose chromatography and crystallization. Inactive mutants such as G446E-hGR or the double mutants G446E/F447P-hGR and G446P/F447P-hGR were isolated by immunoadsorption chromatography. G446E/F447P-hGR was studied in detail. This mutant behaved like a poorly folded monomeric protein, as indicated by the following properties: absence of the intersubunit disulfide bridge, Cys90-Cys90'; failure to bind FAD; failure to bind NADPH and analogues thereof; a short half-life (< 4 min) in E. coli cells; and high susceptibility to trypsin in vitro. The results suggest that the sequence around G446 can control dimerization as well as domain folding. This is unexpected since the FAD-binding domain and the NADPH-binding domain occur in many different enzymes and have been regarded as autonomous folding units.(ABSTRACT TRUNCATED AT 250 WORDS)
Nordhoff
A
A
Department of Biochemistry, Heidelberg University, Germany.
Bücheler
U S
US
Werner
D
D
Schirmer
R H
RH
D000595:Amino Acid Sequence; D000962:Antimalarials / Q000138:chemical synthesis* / Q000494:pharmacology; D001483:Base Sequence; D002846:Chromatography, Affinity; D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics; D005971:Glutamates; D018698:Glutamic Acid; D005980:Glutathione Reductase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005998:Glycine; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D000962:Antimalarials; D005971:Glutamates; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D018698:Glutamic Acid; D005980:Glutathione Reductase; D005998:Glycine