Xl. Ren et al., MUTAGENESIS OF STRUCTURAL HALF-CYSTINE RESIDUES IN HUMAN THIOREDOXIN AND EFFECTS ON THE REGULATION OF ACTIVITY BY SELENODIGLUTATHIONE, Biochemistry, 32(37), 1993, pp. 9701-9708
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.