Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae
Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae
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DOI：
10.1016/j.jmb.2008.10.055
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发表时间：
2009-01-23
影响因子：
5.6
通讯作者：
Soares Netto, Luis Eduardo
中科院分区：
文献类型：
--
作者：
Discola, Karen Fulan;de Oliveira, Marcos Antonio;Soares Netto, Luis Eduardo
谷氧还蛋白（Grxs）是广泛分布的小的（9-12 kDa）热稳定蛋白。在酿酒酵母中，已经鉴定了七种Grx酶。其中两个（yGrx 1和yGrx 2）是二硫代的，具有保守的Cys-Pro-Tyr-Cys基序。在这里，我们表明，yGrx 2的比活性比yGrx 1高15倍，虽然这两个氧化还原酶共享64%的同一性和85%的相似性，相对于它们的氨基酸序列。通过双底物动力学分析的酶活性的进一步表征表明，yGrx 2具有较低的Km谷胱甘肽和较高的营业额比yGrx 1。为了更好地理解这些生物化学差异，测定了这两种蛋白质和yGrx 2-C30 S突变体的N-末端活性位点半胱氨酸（Cys 27）的pK（a）。由于yGrx 1和yGix 2 Cys 27残基的pK（a）值非常相似，因此这些参数不能解释其比活性之间观察到的差异。因此，yGrx 2在氧化形式和谷胱甘肽混合二硫化物的晶体结构分别在2.05和1.91埃的分辨率下测定。yGrx 2结构与最近确定的yGrx 1结构的比较提供了对它们显着的功能分歧的见解。我们推测，由Ala 23和Glu 52在yGrx 2的活性位点C-末端半胱氨酸攻击N-末端活性位点半胱氨酸和谷胱甘肽分子之间的混合二硫键的能力修改的YGrx 1中的Ser 23和Gln 52的取代。突变研究支持这一假设。yGrx 1和yGrx 2之间观察到的结构和功能差异可能反映了底物特异性的变化。(C)2008爱思唯尔有限公司保留所有权利。
Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower Km for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pK(a) of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pK(a) values of the yGrx1 and yGix2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 angstrom, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity. (C) 2008 Elsevier Ltd. All rights reserved.