Comparative and integrative metabolomics reveal that S-nitrosation inhibits physiologically relevant metabolic enzymes

Abstract

Cysteine <i>S</i>-nitrosation is a reversible post-translational modification mediated by nitric oxide (^•NO)-derived agents. <i>S</i>-Nitrosation participates in cellular signaling and is associated with several diseases such as cancer, cardiovascular diseases, and neuronal disorders. Despite the physiological importance of this nonclassical ^•NO-signaling pathway, little is understood about how much <i>S</i>-nitrosation affects protein function. Moreover, identifying physiologically relevant targets of <i>S</i>-nitrosation is difficult because of the dynamics of transnitrosation and a limited understanding of the physiological mechanisms leading to selective protein <i>S</i>-nitrosation. To identify proteins whose activities are modulated by <i>S</i>-nitrosation, we performed a metabolomics study comparing WT and endothelial nitric-oxide synthase knockout mice. We integrated our results with those of a previous proteomics study that identified physiologically relevant <i>S</i>-nitrosated cysteines, and we found that the activity of at least 21 metabolic enzymes might be regulated by <i>S</i>-nitrosation. We cloned, expressed, and purified four of these enzymes and observed that <i>S</i>-nitrosation inhibits the metabolic enzymes 6-phosphogluconate dehydrogenase, Δ1-pyrroline-5-carboxylate dehydrogenase, catechol-<i>O</i>-methyltransferase, and d-3-phosphoglycerate dehydrogenase. Furthermore, using site-directed mutagenesis, we identified the predominant cysteine residue influencing the observed activity changes in each enzyme. In summary, using an integrated metabolomics approach, we have identified several physiologically relevant <i>S</i>-nitrosation targets, including metabolic enzymes, which are inhibited by this modification, and we have found the cysteines modified by <i>S</i>-nitrosation in each enzyme.