Mohr S, Stamler J S, Brüne B
University of Konstanz, Faculty of Biology, Germany.
FEBS Lett. 1994 Jul 18;348(3):223-7. doi: 10.1016/0014-5793(94)00596-6.
Previous studies have suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO.-induced [32P]NAD(+)-dependent mechanism. However, the efficacy of GAPDH modification induced by various NO donors was found to be independent of spontaneous rates of NO. release. To further test the validity of this mechanism, we studied the effects of nitrosonium tertrafluoroborate (BF4NO), a strong NO+ donor. BF4NO potently induces GAPDH labeling by the radioactive nucleotide. In this case, the addition of thiol significantly attenuates enzyme modification by competing for the NO moiety in the formation of RS-NO. Peroxynitrite (ONOO-) also induces GAPDH modification in the presence of thiol, consistent with the notion that this species can transfer NO+ (or NO2+) through the intermediacy of RS-NO. However, the efficiency of this reaction is limited by ONOO- -induced oxidation of protein SH groups at the active site. ONOO- generation appears to account for the modification of GAPDH by SIN-1. Thus, S-nitrosylation of the active site thiol is a prequisite for subsequent post-translational modification with NAD+, and emphasizes the role of NO+ transfer in the initial step of this pathway. Our findings thus provide a uniform mechanism by which nitric oxide and related NO donors initiate non-enzymatic ADP-ribosylation (like) reactions. In biological systems, endogenous RS-NO are likely to support the NO group transfer to thiol-containing proteins.
先前的研究表明，甘油醛-3-磷酸脱氢酶（GAPDH）通过一种一氧化氮（NO）诱导的、依赖于[32P]NAD（+）的机制，对活性位点硫醇进行共价修饰。然而，发现各种NO供体诱导的GAPDH修饰效率与NO的自发释放速率无关。为了进一步验证该机制的有效性，我们研究了强NO+供体四氟硼酸硝鎓（BF4NO）的作用。BF4NO能有效地通过放射性核苷酸诱导GAPDH标记。在这种情况下，添加硫醇通过在RS-NO形成过程中竞争NO部分，显著减弱了酶的修饰。在有硫醇存在的情况下，过氧亚硝酸盐（ONOO-）也能诱导GAPDH修饰，这与该物质可通过RS-NO中间体转移NO+（或NO2+）的观点一致。然而，该反应的效率受到活性位点蛋白质SH基团被ONOO-诱导氧化的限制。SIN-1对GAPDH的修饰似乎是由ONOO-的生成引起的。因此，活性位点硫醇的S-亚硝基化是随后用NAD+进行翻译后修饰的先决条件，并强调了NO+转移在该途径初始步骤中的作用。我们的研究结果因此提供了一种统一的机制，通过该机制一氧化氮及相关NO供体引发非酶促ADP-核糖基化（类似）反应。在生物系统中，内源性RS-NO可能支持NO基团向含硫醇蛋白质的转移。
