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首页> 外文期刊>Biochemistry >Modification of aldose reductase by S-nitrosoglutathione
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Modification of aldose reductase by S-nitrosoglutathione

机译：S-亚硝基谷胱甘肽对醛糖还原酶的修饰

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摘要

Kinetic and structural changes in recombinant human aldose reductase (AR) due to modification by S-nitrosoglutathione (GSNO) were investigated. Incubation of the enzyme with 10-50 microM GSNO led to a time- and concentration-dependent inactivation of the enzyme, with a second-order rate constant of 0.087 +/- 0.009 M-1 min-1. However, upon exhaustive modification, 30-40% of the enzyme activity was retained. The non-inactivated enzyme displayed a 2-3-fold change in Km for NADPH and Km fordl-glyceraldehyde, whereas the Km for the lipid peroxidation product, 4-hydroxy-2-trans nonenal (HNE), was comparable to that of the untreated enzyme. The residual activity of the enzyme after GSNO treatment was less sensitive to inhibition by the active site inhibitor sorbinil or to activation by sulfate. Significantly higher catalytic activity was retained when the enzyme was modified in the presence of NADPH, suggesting relatively low reactivity of the E-NADPH complex with GSNO. The modification site was identified using site-directed mutants in which each of the solvent-exposed cysteines of the enzyme was replaced individually by serine. The mutant C298S was insensitive to GSNO, whereas the sensitivity of the mutants C303S and C80S was comparable to that of the wild-type enzyme. Electrospray ionization mass spectroscopy of the GSNO-modified enzyme revealed a major modified species (70% of the protein) with a molecular mass that was 306 Da higher than that of the untreated enzyme, which is consistent with the addition of a single glutathione molecule to the enzyme. The remaining 30% of the protein displayed a molecular mass that was not significantly different from that of the native enzyme. No nitrosated forms of the enzyme were observed. These results suggest that inactivation of AR by GSNO is due to the selective formation of a single mixed disulfide between glutathione and Cys-298 located at the NADP(H)-binding site of the enzyme.
机译：研究了由于S-亚硝基谷胱甘肽（GSNO）修饰而导致的重组人醛糖还原酶（AR）的动力学和结构变化。用10-50 microM GSNO孵育酶会导致酶的时间和浓度依赖性失活，其二级速率常数为0.087 +/- 0.009 M-1 min-1。但是，经过详尽的修饰，保留了30-40％的酶活性。对于NADPH和Km fordl-甘油醛，未灭活的酶的Km表现出2-3-倍的变化，而脂质过氧化产物4-羟基-2-反式壬醛（HNE）的Km可与NDPPH的Km相比。未经处理的酶。 GSNO处理后，酶的残留活性对活性位点抑制剂山梨醇的抑制作用或对硫酸盐的活化作用较不敏感。当在NADPH存在下对酶进行修饰时，保留了较高的催化活性，这表明E-NADPH复合物与GSNO的反应性相对较低。使用定点突变体鉴定修饰位点，其中酶的每个溶剂暴露的半胱氨酸分别被丝氨酸取代。突变体C298S对GSNO不敏感，而突变体C303S和C80S的敏感性与野生型酶相当。 GSNO修饰的酶的电喷雾电离质谱表明，主要修饰的物种（占蛋白质的70％）的分子量比未处理的酶高306 Da，这与向其中添加一个谷胱甘肽分子相一致。酶。其余30％的蛋白质的分子量与天然酶没有明显差异。没有观察到酶的亚硝化形式。这些结果表明，GSNO使AR失活是由于在谷胱甘肽和位于酶的NADP（H）结合位点的Cys-298之间选择性地形成了一个混合的二硫键。

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