A MIXED SELENODISULPHIDE BOND AT THE ACTIVE SITE OF THIOREDOXIN REDUCTASE

哺乳动物胞质硫氧还蛋白还原酶（TrxR）在黄素环附近有一个对氧化还原敏感的Cys59和Cys64对，在cooh末端附近有一个Cys497和Secys498对。为了表征氧化TrxR中Secys残留物的化学性质，用H2O2或氧化硫氧还蛋白处理TrxR。然后用生物素偶联的碘乙酰胺和碘乙酰胺标记酶，用Lys-C裂解酶，最后用LC/MS分析酶解后的肽混合物。总离子色谱的峰质量为1297.7道尔顿（保留时间为20.5 min），这与含有Cys497和Secys498（用一个字母密码子U表示）的COOH末端片段R487SGGDILQSGCUG499的1297.3道尔顿（由混合硒二硫键（S-Se肽）桥接）的期望峰质量基本相同。Edman降解20.5 min肽得到RSGGDILQSG的完整序列。结合大量数据，测序结果最终证明20.5 min肽确实是S-Se肽。在TrxS2含量有限的情况下，活性位点Cys-SH/Cys-SH对比Cys-SH/Secys-SeH对优先被氧化。TrxS2或H2O2对Cys- sh /Cys- sh对的氧化作用和NADPH对Cys/Cys二硫化物的还原作用均被烷基化抑制。此外，Secys修饰完全阻断了TrxR在NADPH存在下催化TrxS2还原的能力。这些结果表明，Cys/Secys对的功能可能是将活性位点Cys- sh /Cys- sh对的还原等效物传递给TrxS2所必需的。-硫氧还蛋白;硫氧还蛋白还原酶;硒，硫化硒键

Mammalian cytosolic thioredoxin reductase (TrxR) has a redox-sensitive Cys59 and Cys64 pair adjacent to the flavin ring and a Cys497 and Secys498 pair near the COOH-terminus. To characterize the chemical nature of the Secys residue in oxidized TrxR, TrxR was treated with H2O2 or oxidized thioredoxin.. The resulting enzymes were then labeled with biotin-conjugated iodoacetamide followed by iodoacetamide and cleaved with Lys-C, and finally, the digested peptide mixtures were analyzed by LC/MS. Total ion chromatograms included peaks with a mass of 1297.7 daltons (retention time, 20.5 min) which is essentially identical, respectively, to the 1297.3 daltons expected for the COOH- terminal fragment R487SGGDILQSGCUG499 containing Cys497 and Secys498 (denoted by one-letter codon U) bridged by a mixed selenodisulfide bond (S-Se peptide). Edman degradation of the 20.5-min peptide yielded a complete sequence of RSGGDILQSG. Together with the mass data, the sequencing result conclusively proves that the 20.5-min peptide is indeed the S-Se peptide. In the presence of limited amounts of TrxS2, the active site Cys-SH/Cys-SH pair was preferentially oxidized over the Cys-SH/Secys-SeH pair. Both the oxidation of the Cys-SH/Cys-SH pair by TrxS2 or H2O2 and the reduction of the Cys/Cys disulfide by NADPH were inhibited by alkylation of the Secys residue. Furthermore, the Secys modification completely blocked the ability of TrxR to catalyze the reduction of TrxS2 in the presence of NADPH. These results suggest that the function of the Cys/Secys pair may be essential to pass reducing equivalents from the active site Cys-SH/Cys-SH pair to TrxS2 . - thioredoxin; thioredoxin reductase; slenium, selenosulphide bond