Zn(II)-free dimethylargininase-1 (DDAH-1) is inhibited upon specific Cys-S-nitrosylation.
The endogenous nitric oxide synthase inhibitors L-N(omega)-methylarginine and L-N(omega),N(omega)-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.
Knipp
Markus
M
Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. mknipp@bioc.unizh.ch
Braun
Oliver
O
Gehrig
Peter M
PM
Sack
Ragna
R
Vasák
Milan
M
D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D003545:Cysteine / Q000378:metabolism*; D006358:Hot Temperature; D016023:Integrins / Q000502:physiology; D007700:Kinetics; D008024:Ligands; D008322:Mammals; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology; D015538:Nitrosation; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology*
D016023:Integrins; D008024:Ligands; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009569:Nitric Oxide; D000581:Amidohydrolases; C059999:dimethylargininase; D015032:Zinc; D003545:Cysteine