Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7
Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7
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DOI：
10.1016/j.bbagen.2013.02.017
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发表时间：
2013-06-01
影响因子：
3
通讯作者：
Maiorino, Matilde
中科院分区：
文献类型：
--
作者：
Bosello-Travain, Valentina;Conrad, Marcus;Maiorino, Matilde
背景：哺乳动物GPx 7是内质网（ER）的一种单体谷胱甘肽过氧化物酶，含有一个Cys氧化还原中心（CysGPx）。虽然含有一个过氧化物半胱氨酸（CP），它缺乏解决半胱氨酸（CR），赋予快速反应性与硫氧还蛋白（Trx）或相关蛋白质的大多数其他CysGPxs.Methods：减少底物特异性和机制的稳态动力学分析野生型或突变的小鼠GPx 7。将酶异源表达为突触核蛋白融合体以克服有限的表达。磷脂过氧化氢是氧化底物。酶底物。结果：C-P氧化速度快（k（+1）> 10（3）M-1 s（-1）），而GSH还原速度慢（k '（+2）= 12.6 M-1 s（-1）），尽管分子对接表明GSH-GPx 7有很强的相互作用。相反，氧化的C-P可以被人蛋白质二硫键异构酶（HsPDI）以快速速率还原（k（+1）> 10（3）M-1 s（-1）），但不能被Trx还原。通过表面等离子体共振分析，计算出PDI-GPx 7复合物的K-D = 5.2 μ M。排除了另一种非典型的C-R参与过氧化反应的可能性。在生理还原底物浓度的存在下的比活性测量，表明底物竞争在vivo.Conclusions：GPx 7是一个不寻常的CysGPx催化过氧化循环的一个半胱氨酸的机制，其中GSH和PDI是替代substrate.General意义：在ER中，GPx 7的新兴的生理作用是氧化的PDI，调制的GSH的量。(C)2013爱思唯尔有限公司版权所有。
Background: Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C-P) it lacks the resolving Cys (C-R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.Methods: Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme-substrate. and protein-protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.Results: Oxidation of the C-P is fast (k(+1) > 10(3) M-1 s(-1)), however the rate of reduction by GSH is slow (k'(+2) = 12.6 M-1 s(-1)) even though molecular docking indicates a strong GSH-GPx7 interaction. Instead, the oxidized C-P can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k(+1) > 10(3) M-1 s(-1)), but not by Trx. By surface plasmon resonance analysis, a K-D = 5.2 mu M was calculated for PDI-GPx7 complex. Participation of an alternative non-canonical C-R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.Conclusions: GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.General significance: In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH. (C) 2013 Elsevier B.V. All rights reserved.