| Nitric oxide (NO) is a ubiquitous cell-signaling molecule with diverse physiological roles. NO exerts its biological effects by chemical modification of transition metals and cysteine thiols on proteins. The reaction of NO with protein thiols is termed S-nitrosylation and it has been shown to modulate activities of a broad spectrum of proteins with exquisite specificity. In this study, a key enzyme for arginine synthesis is shown to be regulated by S-nitrosylation. The substrate for NO synthesis, L-arginine, can be regenerated from the NOS co-product L-citrulline by the sequential action of two enzymes, argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). AS activity has been shown to be rate-limiting for high-output NO synthesis by immunostimulant-activated cells, and represents a potential site for metabolic control of NO synthesis. Here we demonstrate that NO mediates reversible S-nitrosylation and inactivation of AS in vitro and in vivo. Using a novel mass spectrometry-based method, we show that Cys132 in human AS is the sole target for S-nitrosylation. Mutagenesis studies confirm that S-nitrosylation of Cys132 is both necessary and sufficient for inhibition of AS by NO-donors. S-nitrosylated AS content is shown to be modulated by cellular GSH levels and regulates NO production from citrulline. Phylogenetic comparison of AS homologs, suggests that S-nitrosylation of Cys132 has evolved as a feedback regulatory mechanism to limit NO production by restricting arginine bioavailability. A mechanism of inactivation of AS by S-nitrosylation of Cys132 is proposed based on homology modeling of human AS. Finally, as an extension to the study of AS S-nitrosylation, a proteomic approach was developed for unbiased identification of S-nitrosylated cysteine residues on proteins from cell lysates. Using this method, more than a hundred novel S-nitrosylated cysteines have been identified in mouse tissue lysates. Future bioinformatic analyses are expected to yield consensus motifs for specific S-nitrosylation of cysteines in proteins. |
