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.
Hao
Gang
G
Department of Pharmacology, Weill Medical College of Cornell University, New York, NY 10021, USA.
Xie
Linjun
L
Gross
Steven S
SS
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000473:pathology; D001120:Arginine / Q000737:chemistry; D001124:Argininosuccinate Synthase / Q000037:antagonists & inhibitors* / Q000502:physiology*; D015153:Blotting, Western; D019328:Buthionine Sulfoximine / Q000737:chemistry; D002384:Catalysis; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D009130:Muscle, Smooth / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009211:Myoglobin / Q000737:chemistry; D009569:Nitric Oxide / Q000737:chemistry; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D009584:Nitrogen / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors
D008070:Lipopolysaccharides; D009211:Myoglobin; D020030:Nitric Oxide Donors; D011994:Recombinant Proteins; D009569:Nitric Oxide; D019328:Buthionine Sulfoximine; C042431:S-ethyl glutathione; D001120:Arginine; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D005982:Glutathione Transferase; D001124:Argininosuccinate Synthase; D005978:Glutathione; D003545:Cysteine; D009584:Nitrogen