Argininosuccinate synthetase is reversibly inactivated by S-nitrosylation in vitro and in vivo

Prior studies have demonstrated that the substrate for NO synthesis, L-arginine, can be regenerated from the NOS co-product L-citrulline. This requires 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. We now demonstrate that NO mediates reversible S-nitrosylation and inactivation of AS in vitro and in lipopolysaccharide-treated cells and mice. Using a novel mass spectrometry-based method, we show that Cys-132 in human AS is the sole target for S-nitrosylation among five Cys residues. Mutagenesis studies confirm that S-nitrosylation of Cys-132 is both necessary and sufficient for the inhibition of AS by NO donors. S-nitroso-AS content is regulated by cellular glutathione levels and selectively influences NO production when citrulline is provided to cells as a protosubstrate of NOS but not when L-arginine is provided. A phylogenetic comparison of AS sequences suggests that Cys-132 evolved as a site for post-translational regulation of activity in the AS in NOS-expressing species, endowing NO with the capacity to limit its own synthesis by restricting arginine availability.