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

Abstract The endogenous nitric oxide synthase inhibitorsl-N ω-methylarginine andl-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.