Abstract
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.
Highlights
Nitric oxide (NO)1 is a cell signaling molecule with diverse and important biological functions [1, 2]
A phylogenetic comparison of argininosuccinate synthetase (AS) sequences suggests that Cys-132 evolved as a site for post-translational regulation of activity in the AS in NO synthase (NOS)-expressing species, endowing NO with the capacity to limit its own synthesis by restricting arginine availability
AS and argininosuccinate lyase (AL) are generally considered in the context of their contribution to the urea cycle of the liver, where AS serves as the rate-limiting enzyme for ammonia detoxification [7], these enzymes endow isoform of nitric-oxide synthase (iNOS)-expressing cells with an Arg/Cit cycle for continuous regeneration of Arg from Cit, providing iNOS with a sustained supply of substrate
Summary
Nitric oxide (NO)1 is a cell signaling molecule with diverse and important biological functions [1, 2]. We previously demonstrated that AS activity is rate-limiting for immunostimulant-induced high output NO production in cultured vascular smooth muscle cells [8], and the Arg/Cit cycle provides the preferred source of Arg to iNOS [13].
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