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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Mammalian thioredoxin reductase: Oxidation of the C-terminaI cvsteine/selenocysteine active site forms cyteine/selenocysteine active site forms a thioselenide, and replacement of selenium with sulfur markedly reduces catalytic activity
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Mammalian thioredoxin reductase: Oxidation of the C-terminaI cvsteine/selenocysteine active site forms cyteine/selenocysteine active site forms a thioselenide, and replacement of selenium with sulfur markedly reduces catalytic activity

机译：哺乳动物硫氧还蛋白还原酶：C末端半胱氨酸/硒代半胱氨酸活性位点的氧化形成胱氨酸/硒代半胱氨酸活性位点，形成硫代硒化物，用硫替代硒会明显降低催化活性

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Mammalian Cytosolic thioredoxin reductase (trxR) has a redox center, consisting of Cys~59/Cys~64 adjacent to the flavin ring of FAD and another center consisting of Cys~497/selenocysteine (SeCys)~498 near the C terminus. We now show that the C-terminal Cys~497-SH/SeCys~498- Se-- of NADPH-reduced enzyme, affer anaerobic dialysis, was con- verted to a thioselenide on incubation with excess oxidized Trx rtrxS2) or H_2O_2. The Cys~59-SH/Cys~64-SH pair also was oxidized to a disulfide. At lower concentrations of TrxS~2, the Cys~59-SH/Cys~64-SH center was still converted to a disulfide, presumably by reduction of the thiose- lenide to Cys~497-SH/SeCys~498-Se--. Specific alkylation of SeCys~496 com- pletely blocked the TrxS_2-induced oxidation of Cys~59-SH/Cys~64-SH, and the alkylated enzyme had negligible NADPH-disulfide oxi- doreductase activity. The effect of replacing SeCys~498 with Cys was determined by using a mutant form of human placental TrxR1 expressed in EsCherhoia coli. The NADPH-disulfide oxidoreductase activity of the purified Cys~497/Cys~498 mutant enzyme was 6/100 or 11/100 of that of wiId-type rat liver TrxR1 with 5,5'-dithiobis(2-nitrobenzoic acid) or TrxS~2, respectively, as substrate. Disulfide formation induced by excess TrxS_2 in the mutant form was 12/100 of that of the wild type. Thus, Secys has a critical redox function during the catalytic cyde, which is performed poorly by Cys.
机译：哺乳动物的胞质硫氧还蛋白还原酶（trxR）的氧化还原中心由与FAD黄素环相邻的Cys〜59 / Cys〜64组成，在C末端附近的另一个中心由Cys〜497 /硒代半胱氨酸（SeCys）〜498组成。现在我们显示，厌氧渗析后，NADPH还原酶的C端Cys〜497-SH / SeCys〜498-Se--被转化为硫代硒化物，与过量的氧化Trx rtrxS2）或H_2O_2一起孵育。 Cys〜59-SH / Cys〜64-SH对也被氧化成二硫化物。在较低的TrxS〜2浓度下，Cys〜59-SH / Cys〜64-SH中心仍会转化为二硫化物，大概是通过将硫代硒化物还原为Cys〜497-SH / SeCys〜498-Se-- 。 SeCys〜496的特定烷基化完全阻断了TrxS_2诱导的Cys〜59-SH / Cys〜64-SH的氧化，并且烷基化酶的NADPH-二硫键氧化还原酶活性可忽略不计。通过使用在大肠杆菌中表达的人胎盘TrxR1的突变形式来确定用Cys替代SeCys〜498的效果。纯化的Cys〜497 / Cys〜498突变酶的NADPH-二硫键氧化还原酶活性是具有5,5'-二硫代双（2-硝基苯甲酸）或TrxS的wiId型大鼠肝脏TrxR1的6/100或11/100 〜2，分别作为底物。突变形式的过量TrxS_2诱导的二硫键形成是野生型的12/100。因此，Secys在催化循环中具有关键的氧化还原功能，而Cys的性能很差。

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