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首页> 外文期刊>Biochemistry >Methaneseleninic Acid Is a Substrate for Truncated Mammalian Thioredoxin Reductase: Implications for the Catalytic Mechanism and Redox Signaling
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Methaneseleninic Acid Is a Substrate for Truncated Mammalian Thioredoxin Reductase: Implications for the Catalytic Mechanism and Redox Signaling

机译：甲亚硒酸是截断的哺乳动物硫氧还蛋白还原酶的底物：对催化机制和氧化还原信号的意义。

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Mammalian thioredoxin reductase is a homodimeric pyridine nucleotide disulfide oxidoreductasenthat contains the rare amino acid selenocysteine (Sec) on a C-terminal extension. We previously have shownnthat a truncated version of mouse mitochondrial thioredoxin reductase missing this C-terminal tail willncatalyze the reduction of a number of smallmolecules.Here we show that the truncated thioredoxin reductasenwill catalyze the reduction of methaneseleninic acid. This reduction is fast at pH 6.1 and is only 4-fold slowernthan that of the full-length enzyme containing Sec. This finding suggested to us that if the C-terminal Secnresidue in the holoenzyme became oxidized to the seleninic acid form (Sec-SeO2n-) that it would be quicklynreduced back to an active state by enzymic thiols and further suggested to us that the enzyme would be verynresistant to irreversible inactivation by oxidation. We tested this hypothesis by reducing the enzyme withnNADPH and subjecting it to high concentrations of H2O2 (up to 50 mM). The results show that the enzymenstrongly resisted inactivation by 50 mMH2O2. To determine the redox state of the C-terminal Sec residue, wenattempted to inhibit the enzyme with dimedone. Dimedone alkylates protein sulfenic acid residues andnpresumably will alkylate selenenic acid (Sec-SeOH) residues as well. The enzyme was not inhibited byndimedone even when a 150-fold excess was added to the reaction mixture containing the enzyme and H2O2.nWe also tested the ability of the truncated enzyme to resist inactivation by oxidation as well and found that itnalso was resistant to high concentrations of H2O2. One assumption for the use of Sec in enzymes is that it isncatalytically superior to the use of cysteine.We and others have previously suggested that there are reasons fornthe use of Sec in enzymes that are unrelated to the conversion of substrate to product. The data presented herensupport this assertion. The results also imply that the redox signaling function of the thioredoxin system cannremain active under oxidative stress.
机译：哺乳动物的硫氧还蛋白还原酶是一种同型二聚吡啶核苷酸二硫化物氧化还原酶，在C端延伸部分包含稀有氨基酸硒代半胱氨酸（Sec）。先前我们已经证明，缺失此C末端尾部的小鼠线粒体硫氧还蛋白还原酶的截短形式将催化许多小分子的还原。在这里，我们表明截短的硫氧还蛋白还原酶将催化甲烷硒酸的还原。在pH 6.1时，这种还原速度很快，并且仅比包含Sec的全长酶的还原速度慢4倍。这一发现向我们表明，如果全酶中的C端Secnresidue氧化为硒酸形式（Sec-SeO2n-），它会被酶硫醇迅速还原成活性状态，并进一步向我们表明，该酶会对氧化引起的不可逆失活具有极强的抵抗力。我们通过用nNADPH还原酶并使其经受高浓度的H2O2（最高50 mM）来检验该假设。结果表明，该酶强烈抵抗50 mMH2O2的失活。为了确定C-末端Sec残基的氧化还原状态，尝试用二甲酮抑制该酶。二甲酮使蛋白质亚磺酸残基烷基化，并且大概也将硒酸（Sec-SeOH）残基烷基化。即使向含有该酶和H2O2的反应混合物中加入150倍过量，该酶也不会被苯二甲酮抑制.n我们还测试了截短的酶也具有抗氧化灭活的能力，发现它也能抵抗高浓度H2O2。在酶中使用Sec的一个假设是它在催化上优于半胱氨酸。我们和其他人先前已经提出，在酶中使用Sec的原因与底物向产物的转化无关。此处提供的数据支持此断言。该结果还暗示了硫氧还蛋白系统的氧化还原信号功能在氧化应激下不能保持活性。

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