Chae H Z, Uhm T B, Rhee S G
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7022-6. doi: 10.1073/pnas.91.15.7022.
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.
来自酵母的硫醇特异性抗氧化剂（TSA）在氨基酸位置47和170处含有半胱氨酸残基，但与明显的氧化还原辅因子无关。这两个半胱氨酸在与TSA具有23 - 98%序列同一性的蛋白质家族中高度保守。通过分别用丝氨酸替换酵母TSA中的Cys - 47和Cys - 170，并在大肠杆菌中表达突变型TSA蛋白（分别为RC47S和RC170S）以及野生型TSA（RWT），研究了Cys - 47和Cys - 170在酵母TSA中的作用。从酵母中纯化的野生型TSA（YWT）和RWT均主要以二聚体形式存在，而RC47S和RC170S在变性条件下主要以单体形式存在。这一观察结果表明，YWT和RWT的二聚化需要Cys - 47和Cys - 170的二硫键连接。通过分离二聚体胰蛋白酶肽段直接证明了YWT中存在Cys - 47 - Cys - 170连接，其中一个单体含有Cys - 47，另一个含有Cys - 170。一小部分YWT、RWT、RC47S和RC170S分子形成了由Cys - 47 - Cys - 47或Cys - 170 - Cys - 170二硫键连接的二聚体。根据各种TSA蛋白保护谷氨酰胺合成酶免受二硫苏糖醇/Fe³⁺/O₂氧化系统影响的能力，评估了它们的抗氧化活性。YWT、RWT和RC170S具有同等的保护作用，而RC47S则完全无效。因此，是Cys - 47而非Cys - 170构成了假定底物的氧化位点。
