Bosello-Travain Valentina, Conrad Marcus, Cozza Giorgio, Negro Alessandro, Quartesan Silvia, Rossetto Monica, Roveri Antonella, Toppo Stefano, Ursini Fulvio, Zaccarin Mattia, Maiorino Matilde
Department of Molecular Medicine, University of Padova, Padua, Italy.
Biochim Biophys Acta. 2013 Jun;1830(6):3846-57. doi: 10.1016/j.bbagen.2013.02.017. Epub 2013 Feb 27.
Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.
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.
Oxidation of the CP is fast (k+1>10(3)M(-1)s(-1)), however the rate of reduction by GSH is slow (k'+2=12.6M(-1)s(-1)) even though molecular docking indicates a strong GSH-GPx7 interaction. Instead, the oxidized CP 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 KD=5.2μM was calculated for PDI-GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.
GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.
In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.
哺乳动物的GPx7是一种内质网（ER）的单体谷胱甘肽过氧化物酶，含有一个半胱氨酸氧化还原中心（CysGPx）。尽管含有一个过氧化物半胱氨酸（CP），但它缺乏解离半胱氨酸（CR），而CR能使大多数其他CysGPx与硫氧还蛋白（Trx）或相关蛋白快速反应。
通过对野生型或突变型小鼠GPx7进行稳态动力学分析，研究其还原底物特异性和机制。为克服表达受限，将这些酶作为与突触核蛋白的融合体进行异源表达。磷脂氢过氧化物为氧化底物。通过分子对接和表面等离子体共振分析来研究酶-底物和蛋白质-蛋白质相互作用。
CP的氧化速度很快（k+1>10(3)M(-1)s(-1)），然而，尽管分子对接表明谷胱甘肽（GSH）与GPx7有很强的相互作用，但GSH的还原速度很慢（k'+2=12.6M(-1)s(-1)）。相反，人蛋白质二硫键异构酶（HsPDI）能快速还原氧化的CP（k+1>10(3)M(-1)s(-1)），而Trx则不能。通过表面等离子体共振分析，计算出PDI-GPx7复合物的解离常数KD=5.2μM。排除了另一种非典型CR参与过氧化物反应的可能性。在生理还原底物浓度下的比活性测量表明，体内存在底物竞争。
GPx7是一种不寻常的CysGPx，通过单一半胱氨酸机制催化过氧化物循环，其中GSH和PDI是替代底物。
在内质网中，GPx7新出现的生理作用是由GSH的量调节的PDI氧化。
