Mohr S, Stamler J S, Brüne B
Faculty of Biology, University of Konstanz, P.O. Box 5560 M612, 78434 Konstanz, Germany.
J Biol Chem. 1996 Feb 23;271(8):4209-14. doi: 10.1074/jbc.271.8.4209.
Nitric oxide (NO)-related activity has been associated with an NAD+-dependent modification of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, the mechanism by which NO effects covalent attachment of nucleotide and its role in regulation of enzyme activity are controversial. Recent studies have shown that S-nitrosylation of GAPDH (Cys149) initiates subsequent modification by the pyridinium cofactor. Here we show that NADH rather than NAD+ is the preferred substrate. Transnitrosation from active site S-nitrosothiol to the reduced nicotinamide ring system appears to facilitate protein thiolate attack on the enzyme-bound cofactor. This results in attachment of the intact NADH molecule. Moreover, we find that S-nitrosylation of GAPDH is responsible for reversible enzyme inhibition, whereas attachment of NADH accounts for irreversible enzyme inactivation. S-Nitrosylation may serve to protect GAPDH from oxidant inactivation in settings of cytokine overproduction and to regulate glycolysis. NADH attachment is more likely to be a pathophysiological event associated with inhibition of gluconeogenesis.
一氧化氮（NO）相关活性与糖酵解酶甘油醛-3-磷酸脱氢酶（GAPDH）的NAD⁺依赖性修饰有关。然而，NO影响核苷酸共价连接的机制及其在酶活性调节中的作用存在争议。最近的研究表明，GAPDH（Cys149）的S-亚硝基化引发了随后吡啶辅酶的修饰。在这里我们表明，NADH而非NAD⁺是首选底物。从活性位点S-亚硝基硫醇到还原型烟酰胺环系统的转亚硝基化似乎促进了蛋白质硫醇盐对酶结合辅酶的攻击。这导致完整的NADH分子附着。此外，我们发现GAPDH的S-亚硝基化导致可逆的酶抑制，而NADH的附着导致不可逆的酶失活。S-亚硝基化可能有助于在细胞因子过度产生的情况下保护GAPDH免受过氧化失活，并调节糖酵解。NADH附着更可能是与糖异生抑制相关的病理生理事件。
