College of Pharmaceutical Science, Jilin University, 1266 Fujin Road, Changchun 130021, China.
J Mol Recognit. 2013 Jan;26(1):38-45. doi: 10.1002/jmr.2241.
Accumulating evidence shows that glutathione peroxidase (GPX, EC.1.11.1.9), one of the most important antioxidant selenoenzymes, plays an essential role in protecting cells and tissues against oxidative damage by catalyzing the reduction of hydrogen peroxide by glutathione. Unfortunately, because of the limited availability and poor stability of GPX, it has not been used clinically to protect against oxidative stress. To overcome these problems, it is necessary to generate mimics of GPX. In this study, we have used directed mutagenesis and the inclusion of a selenocysteine (Sec) insertion sequence to engineer the expression in eukaryotic cells of human glutathione transferase zeta1-1 (hGSTZ1-1) with Sec in the active site (seleno-hGSTZ1-1). This modification converted hGSTZ1-1 into an active GPX and is the first time this has been achieved in eukaryotic cells. The GPX activity of seleno-hGSTZ1-1 is higher than that of GPX from bovine liver, indicating Sec at the active site plays an important role in the determination of catalytic specificity and performance. Kinetic studies revealed that the ping-pong catalytic mechanism of Se-hGSTZ1-1 is similar to that of the natural GPX.
越来越多的证据表明，谷胱甘肽过氧化物酶（GPX，EC.1.11.1.9）作为最重要的抗氧化硒酶之一，通过催化谷胱甘肽还原过氧化氢，在保护细胞和组织免受氧化损伤方面发挥着重要作用。不幸的是，由于 GPX 的可用性有限和稳定性差，它尚未在临床上用于抵御氧化应激。为了克服这些问题，有必要生成 GPX 的模拟物。在这项研究中，我们使用定向诱变和包含硒代半胱氨酸（Sec）插入序列的方法，在真核细胞中表达了具有活性部位 Sec 的人谷胱甘肽转移酶 ζ1-1（hGSTZ1-1）（硒代-hGSTZ1-1）。这种修饰将 hGSTZ1-1 转化为具有活性的 GPX，这是首次在真核细胞中实现。硒代-hGSTZ1-1 的 GPX 活性高于牛肝中的 GPX，表明活性部位的 Sec 在确定催化特异性和性能方面发挥着重要作用。动力学研究表明，Se-hGSTZ1-1 的乒乓催化机制与天然 GPX 相似。
