Synthetic seleno-glutaredoxin 3 analogues are highly reducing oxidoreductases with enhanced catalytic efficiency
Synthetic seleno-glutaredoxin 3 analogues are highly reducing oxidoreductases with enhanced catalytic efficiency
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DOI：
10.1021/ja0661414
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发表时间：
2006-12-27
影响因子：
15
通讯作者：
Dawson, Philip E.
中科院分区：
文献类型：
--
作者：
Metanis, Norman;Keinan, Ehud;Dawson, Philip E.
硒酶在维持细胞氧化还原潜力方面起着核心作用。这些酶在其活性部位具有硒基硫键，可催化过氧化氢、亚硫醚和二硫化物的还原。硒/二硫键交换反应对所有这些酶都是共同的，活性部位的氧化还原电位反映了该反应的正向和反向速率之间的比率。含硒半胱氨酸(SEC)酶的制备在实验上具有挑战性。因此，对硒醇在酶活性部位的动力学作用知之甚少，也没有实验测定蛋白质中硒硫键或二硒化键的氧化还原电位。为了全面评价SEC对氧化还原酶氧化还原电位和氧化还原动力学的影响，化学合成了谷氧还蛋白3(Grx3)及其保守的(CXXC)-C-11-C-14活性中心的三个SEC变体。Grx3、Grx3(C11U)和Grx3(C14U)的氧化还原电位分别为-194、-260和-275 mV。Grx3(C11U-C14U)(-309 mV)和硫氧还蛋白(Trx)(-270 mV)之间的氧化还原平衡位置表明二硒键可能在生物体系中起作用。动力学分析符合这样的假设，即SEC变异体的较低氧化还原电位主要是由于活性部位Se具有较大的亲核性，而不是它在交换反应中作为离开基团或“中心原子”的作用。硒-Grx3类似物的Trx还原速率提高了10(2)-10(4)倍，这表明，与硫化物相比，含硒硫键或二硒化键的氧化还原酶具有生理上相容的氧化还原电位和增强的还原动力学。
Selenoenzymes have a central role in maintaining cellular redox potential. These enzymes have selenenylsulfide bonds in their active sites that catalyze the reduction of peroxides, sulfoxides, and disulfides. The selenol/disufide exchange reaction is common to all of these enzymes, and the active site redox potential reflects the ratio between the forward and reverse rates of this reaction. The preparation of enzymes containing selenocysteine (Sec) is experimentally challenging. As a result, little is known about the kinetic role of selenols in enzyme active sites, and the redox potential of a selenenylsulfide or diselenide bond in a protein has not been experimentally determined. To fully evaluate the effects of Sec on oxidoreductase redox potential and kinetics, glutaredoxin 3 (Grx3) and all three Sec variants of its conserved (CXXC)-C-11-C-14 active site were chemically synthesized. Grx3, Grx3(C11U), and Grx3( C14U) exhibited redox potentials of -194, -260, and -275 mV, respectively. The position of redox equilibrium between Grx3(C11U-C14U) (-309 mV) and thioredoxin (Trx) (-270 mV) suggests a possible role for diselenide bonds in biological systems. Kinetic analysis is consistent with the hypothesis that the lower redox potentials of the Sec variants result primarily from the greater nucleophilicity of the active site selenium rather than its role as either a leaving group or a "central atom" in the exchange reaction. The 10(2)-10(4)-fold increase in the rate of Trx reduction by the seleno-Grx3 analogues demonstrates that oxidoreductases containing either selenenylsulfide or diselenide bonds can have physiologically compatible redox potentials and enhanced reduction kinetics in comparison with their sulfide counterparts.