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 ro les 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 Es cherichia coli. Wild-type TSA purified from yeast (YWT) and RWT were b oth shown to exist predominantly as dimers, whereas RC47S and RC170S e xisted mainly as monomers under a denaturing condition. This observati on suggests that the dimerization of YWT and RWT requires disulfide li nkage of Cys-47 and Cys-170. The presence of the Cys-47-Cys-170 linkag e 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 di ners linked by Cys-47-Cys-47 or Cys-170-Cys-170 disulfide bonds. The a ntioxidant activity of the various TSA proteins was evaluated from the ir ability to protect glutamine synthetase against the dithiothreitol/ Fe3+/O-2 oxidation system. YWT, RWT, and RC170S were equally protectiv e, whereas RC47S was completely ineffective. Thus, Cys-47, but not Cys -170, constitutes the site of oxidation by putative substrate.