Zn(II)-free Dimethylargininase-1 (DDAH-1) Is Inhibited upon Specific Cys-S-Nitrosylation

• Published in: The Journal of biological chemistry 2003-01, Vol.278 (5), p.3410-3416
• Main Authors: Knipp, Markus, Braun, Oliver, Gehrig, Peter M, Sack, Ragna, Vasák, Milan
• Format: Article
• Language: English
• Subjects: Amidohydrolases - antagonists & inhibitors; Amidohydrolases - chemistry; Amidohydrolases - genetics; Amidohydrolases - ultrastructure; Amino Acid Sequence; Animals; Binding Sites; Brain - enzymology; Cattle; Cysteine - metabolism; Hot Temperature; Integrins - physiology; Kinetics; Ligands; Mammals; Models, Molecular; Molecular Sequence Data; Nitric Oxide - metabolism; Nitric Oxide - pharmacology; Nitrosation; Peptide Fragments - chemistry; Protein Structure, Secondary; Pseudomonas aeruginosa - enzymology; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Recombinant Proteins - ultrastructure; Sequence Alignment; Sequence Homology, Amino Acid; Thermodynamics; Zinc - metabolism; Zinc - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M209088200

The endogenous nitric oxide synthase inhibitors l - N ω -methylarginine and l - N ω , N ω -dimethylarginine are catabolized by the enzyme dimethylargininase. Dimethylargininase-1 from bovine brain contains one tightly bound Zn(II) coordinated by two cysteine sulfur and two lighter ligands. Activity measurements showed that only the apo-enzyme is active and that the holo-enzyme is activated by zinc removal. In this work, the effect of NO on dimethylargininase-1 structure and its activity was investigated using 2-( N , N -dimethylamino)-diazenolate-2-oxide as an NO source. The results showed that whereas the holo-form was resistant to S -nitrosylation, the apo-form could be modified. The results of absorption spectroscopy, mass spectrometry, and fluorometric S-NO quantification revealed that two of five cysteine residues reacted with NO yielding cysteine- S -NO. The modification reaction is specific, because by liquid chromatography/mass spectrometry experiments of digested S-NO-dimethylargininase-1, cysteines 221 and 273 could be identified as cysteine-NO. Because Zn(II) protects the enzyme against nitrosation, it is suggested that both cysteines are involved in metal binding. However, specific cysteine- S -NO formation occurred in the absence of a characteristic sequence motif. Based on a structural model of dimethylargininase-1, the activation of both cysteines may be accomplished by the close proximity of charged residues in the tertiary structure of the enzyme.