Investigation of the C-terminal redox center of high-Mr thioredoxin reductase by protein engineering and semisynthesis.
High-molecular weight thioredoxin reductases (TRs) catalyze the reduction of the redox-active disulfide bond of thioredoxin, but an important difference in the TR family is the sequence of the C-terminal redox-active tetrapeptide that interacts directly with thioredoxin, especially the presence or absence of a selenocysteine (Sec) residue in this tetrapeptide. In this study, we have employed protein engineering techniques to investigate the C-terminal redox-active tetrapeptides of three different TRs: mouse mitochondrial TR (mTR3), Drosophila melanogaster TR (DmTR), and the mitochondrial TR from Caenorhabditis elegans (CeTR2), which have C-terminal tetrapeptide sequences of Gly-Cys-Sec-Gly, Ser-Cys-Cys-Ser, and Gly-Cys-Cys-Gly, respectively. Three different types of mutations and chemical modifications were performed in this study: insertion of alanine residues between the cysteine residues of the Cys-Cys or Cys-Sec dyads, modification of the charge at the C-terminus, and altering the position of the Sec residue in the mammalian enzyme. The results show that mTR3 is quite accommodating to insertion of alanine residues into the Cys-Sec dyad, with only a 4-6-fold drop in catalytic activity. In contrast, the activity of both DmTR and CeTR2 was reduced 100-300-fold when alanine residues were inserted into the Cys-Cys dyad. We have tested the importance of a salt bridge between the C-terminus and a basic residue that was proposed for orienting the Cys-Sec dyad of mTR3 for proper catalytic position by changing the C-terminal carboxylate to a carboxamide. The result is an enzyme with twice the activity as the wild-type mammalian enzyme. A similar result was achieved when the C-terminal carboxylate of DmTR was converted to a hydroxamic acid or a thiocarboxylate. Last, reversing the positions of the Cys and Sec residues in the catalytic dyad resulted in a 100-fold loss of catalytic activity. Taken together, the results support our previous model of Sec as the leaving group during reduction of the C-terminus during the catalytic cycle.
Eckenroth
Brian E
BE
Department of Biochemistry, University of Vermont, 89 Beaumont Avenue, Given Laboratory, Room B413, Burlington, Vermont 05405, USA.
Lacey
Brian M
BM
Lothrop
Adam P
AP
Harris
Katharine M
KM
Hondal
Robert J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology; D004136:Dinitrobenzenes / Q000737:chemistry; D004331:Drosophila melanogaster / Q000201:enzymology; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008970:Molecular Weight; D009154:Mutation; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry; D015202:Protein Engineering; D013880:Thioredoxin-Disulfide Reductase / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics
D004136:Dinitrobenzenes; D009195:Peroxidase; D013880:Thioredoxin-Disulfide Reductase; C010936:2,4-dinitrothiocyanatobenzene