Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent protein
Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent protein
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DOI：
10.1021/bi0522495
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发表时间：
2006-02-21
期刊：
影响因子：
2.9
通讯作者：
Winther, JR
中科院分区：
文献类型：
--
作者：
Björnberg, O;Ostergaard, H;Winther, JR
氧化还原敏感的黄色荧光蛋白（rxYFP）含有二硫醇二硫对，在热力学上适合于监测细胞内谷胱甘肽氧化还原电位。已知酵母中的Glutaredoxin 1 （Grx1p）催化氧化还原。rxYFP和谷胱甘肽之间的平衡，在这里，我们产生了两个蛋白的融合，rxYFP- grx1p。与分离的亚基相比，分子内还原等价物的转移使得融合蛋白在与谷胱甘肽反应时具有动力学优势。因此，GSSG的氧化速率提高了3300倍。与GSSG的反应很可能完全通过谷胱甘肽化的中间体进行，而不是通过分子内二硫键的转移。然而，当被H2O2、羟乙基二硫醚或胱氨酸氧化时，glutaredoxin结构域首先反应，然后是一个二硫键向另一个结构域的限制性转移（0.13分钟（-1））。因此，对其他氧化剂的反应性仍然很低，使谷胱甘肽具有几乎绝对的特异性。我们进一步研究了Grx1p活性位点的CPYC -> Cys变体，发现单个Cys变体单独和融合时氧化还原酶活性升高。这不能归因于缺乏对二硫化戊二氧还蛋白的非生产性副反应。相反，与碘乙酰胺较慢的烷基化动力学表明剩余半胱氨酸残基的留基能力较好，这可以解释活性增加的原因。
Redox-sensitive yellow fluorescent protein (rxYFP) contains a dithiol disulfide pair that is thermodynamically suitable for monitoring intracellular glutathione redox potential. Glutaredoxin 1 (Grx1p) from yeast is known to catalyze the redox. equilibrium between rxYFP and glutathione, and here, we have generated a fusion of the two proteins, rxYFP-Grx1p. In comparison to isolated subunits, intramolecular transfer of reducing equivalents made the fusion protein kinetically superior in reactions with glutathione. The rate of GSSG oxidation was thus improved by a factor of 3300. The reaction with GSSG most likely takes place entirely through a glutathionylated intermediate and not through transfer of an intramolecular disulfide bond. However, during oxidation by H2O2, hydroxyethyl disulfide, or cystine, the glutaredoxin domain reacted first, followed by a rate-limiting (0.13 min(-1)) transfer of a disulfide bond to the other domain. Thus, reactivity toward other oxidants remains low, giving almost absolute glutathione specificity. We have further studied CPYC -> CPYS variants in the active site of Grx1p and found that the single Cys variant had elevated oxidoreductase activity separately and in the fusion. This could not be ascribed to the lack of an unproductive side reaction to glutaredoxin disulfide. Instead, slower alkylation kinetics with iodoacetamide indicates a better leaving-group capability of the remaining cysteine residue, which can explain the increased activity.