Site-directed mutagenesis of active site cysteines in human thioredoxin produces competitive inhibitors of human thioredoxin reductase and elimination of mitogenic properties of thioredoxin.

J.E Oblong,M Berggren,P.Y Gasdaska,G Powis

doi:10.1016/s0021-9258(17)32631-5

J.E Oblong, M Berggren + Show 2 more

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https://doi.org/10.1016/s0021-9258(17)32631-5

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Journal: The Journal of biological chemistry	Publication Date: Apr 1, 1994
Citations: 136	License type: cc-by

Affiliation: University of Arizona

Abstract

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.

Highlights

Site-directed Mutagenesisof Active Site Cysteines inHuman Thioredoxin Produces Competitive Inhibitorosf Human Thioredoxin Reductase and Eliminationof Mitogenic Properties of Thioredoxin*
Dox system ubiquitous in all organiTsombse. tter under- 1.6.4.5)(Holmgren, 1985).Two half-reactions comprise the acstandthethiolchemistry of the mammalian thiore- tual sequence of catalytic events of Trx reduction by TR in doxin-thioredoxin reductase redox system, mutantsof which the reduction of the FAD prosthetic group ofTRby human thioredoxin were produced by site-directed mNuA-DPH and electron transfer to active site cysteines in TR
C32S, CSSS, and C32S/CSSS failed to stimulate cell Cys-Gly-His-Cys-Lys,the only differencebeing a proline to hisproliferation, showing that the redox acfotirvme of thi- tidine substitutionin protein disulfide isomerase (for a review oredoxin is necessary foreliciting growth stimulation. see Noiva and Lennarz (1992)).Mutation of proline to histidine in theactive site of E. coli Trx resulted in anincrease in protein disulfideisomerase-likeactivity and, surprisingly, an increased

Summary

Introduction

Site-directed Mutagenesisof Active Site Cysteines inHuman Thioredoxin Produces Competitive Inhibitorosf Human Thioredoxin Reductase and Eliminationof Mitogenic Properties of Thioredoxin*. To each culture was added DIT (3p ~ ) , placental tissue asdescribed previously (Oblonget al., 1993).An insulin PDGF, or varying concentrations of Trx variants, and incubation was reduction assay (Luthman and Holmgren, 1982), which monitors the allowed for the indicated times.

Results

Conclusion