The functional role of selenocysteine (Sec) in the catalysis mechanism of large thioredoxin reductases: Proposition of a swapping catalytic triad including a sec-his-glu state
The functional role of selenocysteine (Sec) in the catalysis mechanism of large thioredoxin reductases: Proposition of a swapping catalytic triad including a sec-his-glu state
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DOI：
10.1002/cbic.200400276
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发表时间：
2005-02-01
期刊：
影响因子：
3.2
通讯作者：
Wessjohann, LA
中科院分区：
文献类型：
--
作者：
Brandt, W;Wessjohann, LA
硫氧还蛋白还原酶催化硫氧还蛋白二硫化物和其他一些氧化的细胞成分的还原，它们是含有一个FAD的同源二聚体蛋白，每个亚基接受一个NADPH作为必需的辅因子。其中一些还原酶在C末端控制一种硒半胱氨酸。根据大鼠硫氧还蛋白还原酶的X射线结构，建立了人硫氧还蛋白还原酶的同源模型，并与硫氧还蛋白对接以模拟活性络合物。在蛋白质结构中可以检测到硒半胱氨酸、组氨酸和谷氨酸之间形成了一种新型的催化三联体。通过DFT(B3LYP，lacv3p**)计算，我们可以证明这种三元化合物的形成对于支持从硒醇到组氨酸的质子转移以稳定硒阴离子是必不可少的，该阴离子能够与硫氧还蛋白的二硫键和催化剂相互作用：还原的二硫键开放。虽然从硒半胱氨酸到组氨酸的简单质子转移在热力学上是不利的，但当谷氨酸的羧酸基稳定形成的咪唑阳离子时，它就变得有利了。用半胱氨酸代替硒半胱氨酸的相同过程需要多4千卡摩尔(-1)的能量，这相当于计算出的平衡位移类似于1000：1或10(3)速率加速：接近实验值的约10(2)倍。这些结果为硫氧还蛋白还原酶的催化机制提供了新的见解，并首次解释了在蛋白质中掺入硒半胱氨酸而不是半胱氨酸残基的优势。
Thioredoxin reductases catalyse the reduction of thioredoxin disulfide and some other oxidised cell constituents, They are homodimeric proteins containing one FAD and accepting one NADPH per subunit as essential cofactors. Some of these reductases contolh a selenocysteine at the C terminus. Based on the X-ray structure of rat thioredoxin reductase, homology models of human thioredoxin reductase were created and subsequently docked to thioredoxin to model the active complex. The formation of a new type of a catalytic triad between selenocysteine, histidine and a glutamate could be detected in the protein structure. By means of DFT (B3LYP, lacv3p**) calculations, we could show that the formation of such a triad is essential to support the proton transfer from selenol to a histidine to stabilise a selenolate anion, which is able to interact with the disulfide of thioredoxin and catalyses: the reductive disulfide opening. Whereas a simple proton transfer, from selenocysteine to histidine is thermodynamically disfavoured by some 18 kcal mol(-1), it becomes favoured when the carboxylic acid group of a glutamate stabilises the formed imidazole cation. An identical process with a cysteine instead of selenocysteine will require 4 kcal mol(-1) more energy, which corresponds to a calculated equilibrium shift of similar to 1000:1 or a 10(3) rate acceleration: a value close to the experimental one of about 10(2) times. These re suits give new insights into the catalytic mechanism of thioredoxin reductase and, for the first time, explain the advantage of the incorporation of a selenocysteine instead of a cysteine residue in a protein.