MUTAGENESIS OF STRUCTURAL HALF-CYSTINE RESIDUES IN HUMAN THIOREDOXIN AND EFFECTS ON THE REGULATION OF ACTIVITY BY SELENODIGLUTATHIONE

A human thioredoxin cDNA was modified to optimize Escherichia coli exp ression and subcloned into the plasmid pACA, a vector for T7 RNA polym erase-directed expression. The substitution of structural (noncatalyti c) 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. Char acterization 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 majo r metabolite of inorganic selenium compounds in mammalian tissues. The K(m) and k(cat), of wild-type hTrx for human placenta thioredoxin red uctase (HP-TR) at pH 7.0 were 2.0 muM and 2800 min-1, respectively. Th e mutant proteins G61S, C72S, and C61S/C72S had K(m) and k(cat), value s similar to those of the wt thioredoxin. Tryptophan fluorescence meas urements 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 g roups. After 3.5 h only 0.5 of initially 4.6 SH groups/thioredoxin rem ained. With the oxidized protein, a pronounced lag phase in thioredoxi n reductase-dependent insulin disulfide reduction was present. Disulfi de-linked dimers of the protein were present. The results clearly show ed that noncatalytic cysteine residues in hTrx were oxidized accompani ed by dimerization and inactivation. The activities of the mutant prot eins 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 C61S hT rx were inhibitors of calf thymus thioredoxin reductase-dependent redu ction of 5,5'-dithiobis(2-nitrobenzoic acid). Substitution of Cys 72 w ith Ser prevented this inhibition. The results show that Cys 72 is cri tical for the specific characteristics of hTrx related to potential re gulation of activity via formation of intra- or intermolecular disulfi des.