Reduction of S-nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry
Reduction of S-nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry
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DOI：
10.1046/j.1432-1033.2003.03486.x
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发表时间：
2003-03-01
期刊：
影响因子：
--
通讯作者：
Höög, JO
中科院分区：
文献类型：
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作者：
Hedberg, JJ;Griffiths, WJ;Höög, JO
人乙醇脱氢酶 3/谷胱甘肽依赖性甲醛脱氢酶被证明能够快速且不可逆地催化 S-亚硝基谷胱甘肽的还原分解。研究了人类乙醇脱氢酶 3 的野生型和两种突变形式的 S-亚硝基谷胱甘肽还原的稳态动力学，这些突变先前已被证明会影响底物 S-羟甲基谷胱甘肽的氧化效率。野生型酶很容易还原 S-亚硝基谷胱甘肽，其 k (cat) /K (m) 约为 S-羟甲基谷胱甘肽氧化的 k (cat) /K (m) 的两倍，从而产生人类乙醇脱氢酶迄今为止确定的最高催化效率。以与S-羟甲基谷胱甘肽氧化类似的方式，通过用Ser或Lys替代Arg115，S-亚硝基谷胱甘肽还原的催化效率显着降低，支持类似的底物结合。 NADH 是迄今为止比 NADPH 更好的辅酶，此前有人认为 NADPH 可以通过低胞质 NADH/NAD(+) 比率来防止乙醇脱氢酶催化的还原反应。然而，通过电喷雾串联质谱法鉴定出S-亚硝基谷胱甘肽还原的主要产物为谷胱甘肽亚磺酰胺和氧化型谷胱甘肽，这两种物质在纯化形式下都不能作为酶的底物或抑制剂。因此，反应产物不是醇脱氢酶3的底物，因此整个反应是不可逆的。我们认为乙醇脱氢酶 3 催化的 S-亚硝基谷胱甘肽还原与人类 NO 代谢具有生理相关性。
Human alcohol dehydrogenase 3/glutathione-dependent formaldehyde dehydrogenase was shown to rapidly and irreversibly catalyse the reductive breakdown of S -nitrosoglutathione. The steady-state kinetics of S -nitrosoglutathione reduction was studied for the wild-type and two mutated forms of human alcohol dehydrogenase 3, mutations that have previously been shown to affect the oxidative efficiency for the substrate S -hydroxymethylglutathione. Wild-type enzyme readily reduces S -nitrosoglutathione with a k (cat) /K (m) approximately twice the k (cat) /K (m) for S -hydroxymethylglutathione oxidation, resulting in the highest catalytic efficiency yet identified for a human alcohol dehydrogenase. In a similar manner as for S -hydroxymethylglutathione oxidation, the catalytic efficiency of S -nitrosoglutathione reduction was significantly decreased by replacement of Arg115 by Ser or Lys, supporting similar substrate binding. NADH was by far a better coenzyme than NADPH, something that previously has been suggested to prevent reductive reactions catalysed by alcohol dehydrogenases through the low cytolsolic NADH/NAD(+) ratio. However, the major products of S -nitrosoglutathione reduction were identified by electrospray tandem mass spectrometry as glutathione sulfinamide and oxidized glutathione neither of which, in their purified form, served as substrate or inhibitor for the enzyme. Hence, the reaction products are not substrates for alcohol dehydrogenase 3 and the overall reaction is therefore irreversible. We propose that alcohol dehydrogenase 3 catalysed S -nitrosoglutathione reduction is of physiological relevance in the metabolism of NO in humans.