Site-directed mutagenesis of active site cysteines in human thioredoxin produces competitive inhibitors of human thioredoxin reductase and elimination of mitogenic properties of thioredoxin.
Thioredoxin and thioredoxin reductase comprise a redox system ubiquitous in all organisms. To better understand the thiol chemistry of the mammalian thioredoxin-thioredoxin reductase redox system, mutants of human thioredoxin were produced by site-directed mutagenesis in which the two active site cysteines were replaced by serine residues, individually (C32S and C35S) and in combination (C32S/C35S). C35S and C32S/C35S were found to be competitive inhibitors of the reduction of human thioredoxin by human thioredoxin reductase with Ki values of 1.8 and 6.7 microM, respectively. C32S did not inhibit thioredoxin reductase, apparently due to aggregation of the oxidized C32S species. Examination of the three mutant forms of thioredoxin by circular dichroism spectroscopy indicated that there were significant differences in the secondary structures when compared with thioredoxin. There were detectable changes in the circular dichroism spectra when thioredoxin, C35S, and C32S/C35S were bound to thioredoxin reductase, whereas C32S with thioredoxin reductase underwent only a small spectral change. Recombinant human thioredoxin stimulated DNA synthesis and the proliferation of murine fibroblasts. The ability of thioredoxin to stimulate cell proliferation could not be duplicated by either dithiothreitol or glutathione. C32S, C35S, and C32S/C35S failed to stimulate cell proliferation, showing that the redox active form of thioredoxin is necessary for eliciting growth stimulation.
Oblong
J E
JE
Arizona Cancer Center, Tucson 85724.
Berggren
M
M
Gasdaska
P Y
PY
Powis
G
G
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D002942:Circular Dichroism; D003545:Cysteine; D004247:DNA / Q000096:biosynthesis; D006801:Humans; D007700:Kinetics; D051379:Mice; D008934:Mitogens / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors*; D013879:Thioredoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology*
D008934:Mitogens; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013879:Thioredoxins; D004247:DNA; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine