Mechanism for the inhibition of aldehyde dehydrogenase by nitric oxide
Mechanism for the inhibition of aldehyde dehydrogenase by nitric oxide
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DOI：
10.1016/s0741-8329(96)00142-5
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发表时间：
1997-03-01
期刊：
影响因子：
2.3
通讯作者：
Nagasawa, HT
中科院分区：
文献类型：
--
作者：
DeMaster, EG;Redfern, B;Nagasawa, HT
气态一氧化氮（NO）和壬酸二乙胺生成的NO对酿酒酵母乙醛脱氢酶（AlDH）的抑制作用具有时间和浓度依赖性。氧的存在下显着降低了NO的抑制程度，表明NO本身，而不是NO的氧化产物，如N2 O3是在生理条件下的抑制物种。基于以下观察结果，酶活性位点处的半胱氨酸残基与这种抑制有关：a）NAD（+）和NADP（+），但不是还原的辅因子，显著增强NO对AlDH的抑制; B）醛底物苯甲醛阻断抑制;和c）抑制伴随着酶上游离巯基的损失。NO失活的酶的活性很容易恢复治疗与二硫苏糖醇（DTT），但不是与GSH。这种差异部分归因于。涉及导致形成环状DTT二硫化物的氧化还原过程。基于从模型系统推导的化学，NO与AlDH巯基的反应被证明产生分子内二硫化物和N2 O。通过对NO抑制酶的非还原SDS-PAGE分析，这些二硫化物被证明是亚基内的二硫化物。在完全抑制AlDH的NO，四个滴定（埃尔曼试剂）sulkydryl组的AlDH被发现被氧化为二硫化物。这些结果表明：a）活性位点Cys-302的巯基和邻近的半胱氨酸被NO氧化形成亚基内二硫化物; B）ALDH的四个亚基中只有两个具有催化活性;和c）NO优先氧化催化活性亚基的巯基。提出了NO抑制AlDH的详细机制。(C)1997年爱思唯尔科学公司
The inhibition of Saccharomyces cerevisiae aldehyde dehydrogenase (AlDH) by gaseous nitric oxide (NO) in solution and by NO generated from diethylamine nonoate was time and concentration dependent. The presence of oxygen significantly reduced the extent of inhibition by NO, indicating that NO itself rather than an oxidation product of NO such as N2O3 is the inhibitory species under physiological conditions. A cysteine residue at the active site of the enzyme was implicated in this inhibition based on the following observations: a) NAD(+) and NADP(+), but not reduced cofactors, significantly enhanced inhibition of AlDH by NO; b) the aldehyde substrate, benzaldehyde, blocked inhibition; and c) inhibition was accompanied by loss of free sulfhydryl groups on the enzyme. Activity of the NO-inactivated enzyme was readily restored by treatment with dithiothreitol (DTT), but not with GSH. This difference was attributed, in part. to a redox process leading to the formation of a cyclic DTT disulfide. Based on the chemistry deduced from model systems, the reaction of NO with AlDH sulfhydryls was shown to produce intramolecular disulfides and N2O. These disulfides were shown to be intrasubunit disulfides by nonreducing SDS-PAGE analysis of the NO-inhibited enzyme. Following complete inhibition of AlDH by NO, four of the eight titratable (Ellman's reagent) sulkydryl groups of AlDH were found to be oxidized to disulfides. These results suggest that a) the sulfhydryl group of active site Cys-302 and a proximal cysteine are oxidized to form an intrasubunit disulfide by NO; b) only two of the four subunits of ALDH art: catalytically active; and c) NO preferentially oxidizes sulfhydryl groups of the catalytically active subunits. A detailed mechanism for the inhibition of AlDH by NO is presented. (C) 1997 Elsevier Science Inc.