Selenium as an electron acceptor during the catalytic mechanism of thioredoxin reductase.
Selenium as an electron acceptor during the catalytic mechanism of thioredoxin reductase.
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DOI：
10.1021/bi400658g
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发表时间：
2014-02-04
期刊：
影响因子：
2.9
通讯作者：
Hondal, Robert J.
中科院分区：
文献类型：
--
作者：
Lothrop, Adam P.;Snider, Greg W.;Ruggles, Erik L.;Patel, Amar S.;Lees, Watson J.;Hondal, Robert J.
哺乳动物硫氧还蛋白还原酶是一种吡啶核苷酸二硫化物氧化还原酶，它在氧化还原活性四肽Gly-Cys-Sec-Gly基序中用稀有的氨基酸硒半胱氨酸(Sec)代替常用的氨基酸半胱氨酸(Cys)来催化硫醇/二硫键交换反应。SEC可以加速这些交换反应的速度：(I)通过比Cys更好的亲核剂，(Ii)通过比Cys更好的亲水性，(Iii)通过比Cys更好的离开基团，或(Iv)通过使用所有这三个因素的组合，比Cys在化学上更活跃。最近，通过创建人硫氧还蛋白还原酶-1的突变体，将C端氧化还原中心的Cys497-Sec498二联体突变为Ser497-Cys498双联体或Cys497-Ser498双联体，证明了亚硒酸酯作为亲核剂在反应机理中的作用。这两种突变酶都与人硫氧还蛋白(Trx)孵育，以确定哪个突变形成了混合的二硫键复合体。只有含有Ser497-Cys498二联体的突变体形成了一个复合体，这种结构已经通过X射线结晶学[Fritz-Wolf，K.，Kehr，S.，Stumpf，M.，Rahlfs，S.和Becker，K.(2011)人硫氧还蛋白还原酶-硫氧还蛋白复合体的晶体结构]来确定。纳特。交警。2383]。这一实验观察很可能意味着，硒是最初攻击TrX的二硫键的亲核试剂，因为只有当Cys存在于二联体的第二位置时，才会产生络合物。作为一种亲核试剂，Sec的亚硒酸盐有助于加快Sec→半胱氨酸突变酶中相对于半胱氨酸的交换反应速度。在酶的酶循环中发生的另一个硫醇/二硫键交换反应是电子从交换中心的硫酸盐转移到C-端氧化还原中心的8元硫代硫环上。硫化硒中的硒原子可以作为良好的离开基团(攻击硫原子)或亲电基团(攻击硒原子)来加速这一交换反应。在这里，我们提供了强有力的证据，证明在这一交换步骤中，硒原子受到了攻击。这是通过创造一种含有Gly-Gly-Seccoo基序的突变酶来说明的，该基序的活性是野生型酶的0.5%。这个突变体缺乏邻近的、可分解的半胱氨酸残基，该残基通过攻击酶和底物之间的混合硫键发挥作用。当SEC被同型半胱氨酸取代时，也得到了类似的结果。这些结果突出了硒作为电子受体在硫氧还蛋白还原酶催化机制中的作用，以及它作为底物电子供体的既定角色。
Mammalian thioredoxin reductase (TR) is a pyridine nucleotide disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys) in the redox-active tetrapeptide Gly-Cys-Sec-Gly motif to catalyze thiol/disulfide exchange reactions. Sec can accelerate the rate of these exchange reactions (i) by being a better nucleophile than Cys, (ii) by being a better electrophile than Cys, (iii) by being a better leaving group than Cys, or (iv) by using a combination of all three of these factors, being more chemically reactive than Cys. The role of the selenolate as a nucleophile in the reaction mechanism was recently demonstrated by creating a mutant of human thioredoxin reductase-1 in which the Cys497-Sec498 dyad of the C-terminal redox center was mutated to either a Ser497-Cys498 dyad or a Cys497-Ser498 dyad. Both mutant enzymes were incubated with human thioredoxin (Trx) to determine which mutant formed a mixed disulfide bond complex. Only the mutant containing the Ser497-Cys498 dyad formed a complex, and this structure has been determined by X-ray crystallography [Fritz-Wolf, K., Kehr, S., Stumpf, M., Rahlfs, S., and Becker, K. (2011) Crystal structure of the human thioredoxin reductase-thioredoxin complex. Nat. Commun. 2, 383]. This experimental observation most likely means that the selenolate is the nucleophile initially attacking the disulfide bond of Trx because a complex resulted only when Cys was present in the second position of the dyad. As a nucleophile, the selenolate of Sec helps to accelerate the rate of this exchange reaction relative to Cys in the Sec → Cys mutant enzyme. Another thiol/disulfide exchange reaction that occurs in the enzymatic cycle of the enzyme is the transfer of electrons from the thiolate of the interchange Cys residue of the N-terminal redox center to the eight-membered selenosulfide ring of the C-terminal redox center. The selenium atom of the selenosulfide could accelerate this exchange reaction by being a good leaving group (attack at the sulfur atom) or by being a good electrophile (attack at the selenium atom). Here we provide strong evidence that the selenium atom is attacked in this exchange step. This was shown by creating a mutant enzyme containing a Gly-Gly-Seccoo- motif that had 0.5% of the activity of the wild-type enzyme. This mutant lacks the adjacent, resolving Cys residue, which acts by attacking the mixed selenosulfide bond that occurs between the enzyme and substrate. A similar result was obtained when Sec was replaced with homocysteine. These results highlight the role of selenium as an electron acceptor in the catalytic mechanism of thioredoxin reductase as well as its established role as a donor of an electron to the substrate.
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影响因子：
3.2
作者：
Brandt, W;Wessjohann, LA
通讯作者：
Wessjohann, LA
影响因子：
2.9
作者：
Gough, JD;Gargano, JM;Lees, WJ
通讯作者：
Lees, WJ
DOI：
10.1039/jr9630001947
发表时间：
1963-01-01
期刊：
JOURNAL OF THE CHEMICAL SOCIETY
影响因子：
--
作者：
GRICE, R;OWEN, LN
通讯作者：
OWEN, LN
影响因子：
4.8
作者：
Cheng, Qing;Sandalova, Tatyana;Arner, Elias S. J.
通讯作者：
Arner, Elias S. J.
影响因子：
8
作者：
Evans, TC;Benner, J;Xu, MQ
通讯作者：
Xu, MQ