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Mutagenesis of structural half-cystine residues in human thioredoxin and effects on the regulation of activity by selenodiglutathione

, : Mutagenesis of structural half-cystine residues in human thioredoxin and effects on the regulation of activity by selenodiglutathione. Biochemistry. 32(37): 9701-9708

A human thioredoxin cDNA was modified to optimize Escherichia coli expression and subcloned into the plasmid pACA, a vector for T7 RNA polymerase-directed expression. The substitution of structural (noncatalytic) half-cystines in human thioredoxin (hTrx) was made by site-directed mutagenesis. The recombinant wild-type (wt) hTrx and its mutants C61S, C72S, and C61S/C72S were expressed and purified to homogeneity. Characterization of the wt and mutant hTrx was done with respect to redox activity with thioredoxin reductase (TR), tryptophan fluorescence, and effects of incubation with GS-Se-SG, which is believed to be the major metabolite of inorganic selenium compounds in mammalian tissues. The K-m and k-cat of wild-type hTrx for human placenta thioredoxin reductase (HP-TR) at pH 7.0 were 2.0 mu-M and 2800 min-1, respectively. The mutant proteins C61S, C72S, and C61S/C72S had K-m and k-cat values similar to those of the wt thioredoxin. Tryptophan fluorescence measurements showed that the wt and mutant proteins had similar stability to a denaturing agent. Incubation of fully reduced thioredoxin with 0.1 molar equivalent of GS-Se-SG resulted in continued oxidation of SH groups. After 3.5 h only 0.5 of initially 4.6 SH groups/thioredoxin remained. With the oxidized protein, a pronounced lag phase in thioredoxin reductase-dependent insulin disulfide reduction was present. Disulfide-linked dimers of the protein were present. The results clearly showed that noncatalytic cysteine residues in hTrx were oxidized accompanied by dimerization and inactivation. The activities of the mutant proteins C72S and C61S/C72S were unchanged after 3 h of incubation with GS-Se-SG. No dimer appeared of the C72S thioredoxin. Both wt and C61Sh Trx were inhibitors of calf thymus thioredoxin reductase-dependent reduction of 5,5'-dithiobis(2-nitrobenzoic acid). Substitution of Cys 72 with Ser prevented this inhibition. The results show that Cys 72 is critical for the specific characteristics of hTrx related to potential regulation of activity via formation of intra- or intermolecular disulfides.


PMID: 8373774

DOI: 10.1021/bi00088a023

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