S-nitrosoglutathione reversibly inhibits GAPDH by S-nitrosylation.
S-nitrosoglutathione reversibly inhibits GAPDH by S-nitrosylation.
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DOI：
10.1152/ajpcell.1995.269.3.c739
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发表时间：
1995-09
期刊：
影响因子：
--
通讯作者：
C. M. Padgett;A. Whorton
中科院分区：
文献类型：
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作者：
C. M. Padgett;A. Whorton
一氧化氮（NO）由血管内皮细胞产生，介导生理和病理反应。尽管导致 NO 介导的内皮细胞损伤的分子靶标尚不清楚，但其中之一是糖酵解酶、3-磷酸甘油醛脱氢酶 (GAPDH)。在本研究中，我们研究了 NO 介导的 GAPDH 抑制的机制，发现 S-亚硝基谷胱甘肽 (GSNO) 抑制纯化酶制剂和内皮细胞中的 GAPDH 活性。此外，GSNO介导的GAPDH抑制是通过修饰GAPDH中的活性位点半胱氨酸残基而发生的，因为与活性位点半胱氨酸残基相互作用的底物甘油醛-3-磷酸浓度的增加保护GAPDH免受GSNO的抑制。尽管在某些条件下，GSNO 和 NO 供体硝普钠 (SNP) 都会导致 GAPDH 的共价 NAD(+) 依赖性修饰，但这种推定的 ADP 核糖基化不太可能是抑制的主要机制，因为化学计量极低，而且就 GSNO 而言，抑制可被硫醇试剂完全逆转。此外，GSNO有效地S-亚硝基化GAPDH，并且亚硝基化程度与抑制程度线性相关，因此每摩尔GAPDH单体需要添加1摩尔NO才能抑制该酶。与这一发现一致的是，GSNO介导的GAPDH抑制作用是可逆的，并且低分子量硫醇可以逆转抑制作用，并且抑制作用的逆转与GAPDH的“脱亚硝基化”相关。这些结果表明，内皮 GAPDH 是 NO 的靶标，并且抑制主要通过 GAPDH 中活性位点半胱氨酸残基的可逆 S-亚硝基化发生。
Nitric oxide (NO), produced by vascular endothelial cells, mediates both physiological and pathological responses. Although the molecular targets responsible for NO-mediated endothelial cell injury are not known, one candidate is the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In this study, we investigated the mechanism involved in NO-mediated GAPDH inhibition and found that S-nitrosoglutathione (GSNO) inhibited GAPDH activity in both purified enzyme preparations and endothelial cells. Furthermore, GSNO-mediated GAPDH inhibition occurred by modification of the active site cysteine residue in GAPDH, since increasing concentrations of the substrate, glyceraldehyde-3-phosphate, which interacts with the active site cysteine residue, protected GAPDH from inhibition by GSNO. Although under certain conditions both GSNO and the NO donor, sodium nitroprusside (SNP), led to the covalent NAD(+)-dependent modification of GAPDH, this putative ADP ribosylation was unlikely to be the primary mechanism for inhibition, since the stoichiometry was extremely low, and, in the case of GSNO, inhibition was completely reversed by thiol reagents. Furthermore, GSNO effectively S-nitrosylated GAPDH, and the extent of nitrosylation was linearly correlated with the degree of inhibition such that addition of 1 mole of NO per mole of GAPDH monomer was necessary to inhibit the enzyme. Consistent with this finding, GSNO-mediated GAPDH inhibition was reversible with low-molecular-weight thiols, and the reversal of inhibition correlated with the "denitrosylation" of GAPDH. These results suggest that endothelial GAPDH is a target for NO and that inhibition occurs principally by the reversible S-nitrosylation of the active site cysteine residue in GAPDH.