Dimerization of thiol-specific antioxidant and the essential role of cysteine 47.
Thiol-specific antioxidant (TSA) from yeast contains cysteine residues at amino acid positions 47 and 170 but is not associated with obvious redox cofactors. These two cysteines are highly conserved in a family of proteins that exhibit sequence identity of 23-98% with TSA. The roles of Cys-47 and Cys-170 in yeast TSA were investigated by replacing them individually with serine and expressing the mutant TSA proteins (RC47S and RC170S, respectively), as well as wild-type TSA (RWT), in Escherichia coli. Wild-type TSA purified from yeast (YWT) and RWT were both shown to exist predominantly as dimers, whereas RC47S and RC170S existed mainly as monomers under a denaturing condition. This observation suggests that the dimerization of YWT and RWT requires disulfide linkage of Cys-47 and Cys-170. The presence of the Cys-47-Cys-170 linkage in YWT was directly shown by isolation of dimeric tryptic peptides, one monomer of which contained Cys-47 and the other contained Cys-170. A small percentage of YWT, RWT, RC47S, and RC170S molecules formed dimers linked by Cys-47-Cys-47 or Cys-170-Cys-170 disulfide bonds. The antioxidant activity of the various TSA proteins was evaluated from their ability to protect glutamine synthetase against the dithiothreitol/Fe3+/O2 oxidation system. YWT, RWT, and RC170S were equally protective, whereas RC47S was completely ineffective. Thus, Cys-47, but not Cys-170, constitutes the site of oxidation by putative substrate.
Chae
H Z
HZ
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Uhm
T B
TB
Rhee
S G
SG
D000595:Amino Acid Sequence; D000975:Antioxidants / Q000737:chemistry*; D001483:Base Sequence; D001704:Biopolymers; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry*; D004271:DNA, Fungal; D004220:Disulfides; D004926:Escherichia coli; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010544:Peroxidases; D054464:Peroxiredoxins; D011506:Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
D000975:Antioxidants; D001704:Biopolymers; D004271:DNA, Fungal; D004220:Disulfides; D011506:Proteins; D010544:Peroxidases; D054464:Peroxiredoxins; D003545:Cysteine