Studies of an active site mutant of the selenoprotein thioredoxin reductase: the Ser-Cys-Cys-Ser motif of the insect orthologue is not sufficient to replace the Cys-Sec dyad in the mammalian enzyme.
We have mutated the redox active C-terminal motif, Gly-Cys-Sec-Gly, of the mammalian selenoprotein thioredoxin reductase (TrxR) to mimic the C-terminal Ser-Cys-Cys-Ser motif of the non-selenoprotein orthologue of Drosophila melanogaster (DmTrxR). The activity of DmTrxR is almost equal to that of mammalian TrxR, which is surprising, because Cys mutants of selenoproteins are normally 1-2 orders of magnitude less active than their selenocysteine (Sec) containing counterparts. It was shown earlier that the flanking Ser residues were important for activating the Cys residues in DmTrxR (Gromer, et.al. (2003) PNAS 100, 12618-12623). However, the "Drosophila mimic" mutant of the mammalian enzyme studied herein had <0.5% activity compared to wild-type. Rapid kinetic studies revealed that all of the redox centers of the mutant were active, but that the C-terminal dithiols were not effective reductants of thioredoxin. The charge-transfer complex of the two-electron reduced enzyme slowly disappeared as the N-terminal dithiols reduced the C-terminal disulfide. In wild-type enzyme, the selenenylsulfide is more difficult to reduce and the charge-transfer complex is more stable. These findings suggest that features in addition to the flanking Ser residues are important for facilitating the high activity of the insect enzyme and that the corresponding features are absent in mammalian TrxR.
Johansson
Linda
L
Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Arscott
L David
LD
Ballou
David P
DP
Williams
Charles H
CH
Arnér
Elias S J
ES
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D017931:DNA Primers; D004331:Drosophila melanogaster; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009154:Mutation; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry*; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D012694:Serine; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine