Human quiescin-sulfhydryl oxidase, QSOX1: Probing internal redox steps by mutagenesis
Human quiescin-sulfhydryl oxidase, QSOX1: Probing internal redox steps by mutagenesis
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DOI：
10.1021/bi702522q
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发表时间：
2008-04-29
期刊：
影响因子：
2.9
通讯作者：
Thorpe, Colin
中科院分区：
文献类型：
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作者：
Heckler, Erin J.;Alon, Assaf;Thorpe, Colin
黄素蛋白静止素-巯基氧化酶（QSOX）快速地将二硫键插入未折叠的、还原的蛋白质中，伴随着氧还原成过氧化氢。本研究报告的第一个异源表达和酶学特性的人QSOX I亚型。与从禽蛋白色中分离的QSOX一样，重组HsQSOX 1对还原型核糖核酸酶A（RNase）和二硫苏糖醇具有高活性，但对还原型谷胱甘肽显示出> 100倍的低k（cat）/K-m。先前对禽类QSOX的研究产生了一种模型，其中还原当量被提议通过酶从第一个硫氧还蛋白结构域（C70-C73）传递到远端二硫化物（C509-C512），然后穿过二聚体界面传递到FAD-近端二硫化物（C449-C452），最后传递到FAD。目前的工作表明，与天然禽酶不同，HsQSOX 1是单体的。该重组表达系统能够构建第一个半胱氨酸突变体，用于该酶家族的机械解剖。突变体HsQSOX 1的活性测定表明，保守的远端C509-C512二硫键被还原的RNA酶或二硫苏糖醇的氧化所取代。选择用于诱变的其他四个半胱氨酸残基C70、C73、C449和C452对于还原的RNA酶的有效氧化都是至关重要的。近端二硫键的C452被证明是QSOX黄素环的电荷转移供体，其配偶体C449被认为是交换硫醇，与硫氧还蛋白结构域中的C70形成混合二硫键。这些数据表明，所有的内部氧化还原步骤发生在哺乳动物QSOX的同一多肽链内，并开始与还原的硫氧还蛋白结构域和Erv/ALR结构域的近端二硫化物之间的直接相互作用。
The flavoprotein quiescin-sulfhydryl oxidase (QSOX) rapidly inserts disulfide bonds into unfolded, reduced proteins with the concomitant reduction of oxygen to hydrogen peroxide. This study reports the first heterologous expression and enzymological characterization of a human QSOX I isoform. Like QSOX isolated from avian egg white, recombinant HsQSOX1 is highly active toward reduced ribonuclease A (RNase) and dithiothreitol but shows a > 100-fold lower k(cat)/K-m for reduced glutathione. Previous studies on avian QSOX led to a model in which reducing equivalents were proposed to relay through the enzyme from the first thioredoxin domain (C70-C73) to a distal disulfide (C509-C512), then across the dimer interface to the FAD-proximal disulfide (C449-C452), and finally to the FAD. The present work shows that, unlike the native avian enzyme, HsQSOX1 is monomeric. The recombinant expression system enabled construction of the first cysteine mutants for mechanistic dissection of this enzyme family. Activity assays with mutant HsQSOX1 indicated that the conserved distal C509-C512 disulfide is dispensable for the oxidation of reduced RNase or dithiothreitol. The four other cysteine residues chosen for mutagenesis, C70, C73, C449, and C452, are all crucial for efficient oxidation of reduced RNase. C452, of the proximal disulfide, is shown to be the charge-transfer donor to the flavin ring of QSOX, and its partner, C449, is expected to be the interchange thiol, forming a mixed disulfide with C70 in the thioredoxin domain. These data demonstrate that all the internal redox steps occur within the same polypeptide chain of mammalian QSOX and commence with a direct interaction between the reduced thioredoxin domain and the proximal disulfide of the Erv/ALR domain.