Dimethylarginine dimethylaminohydrolase (DDAH) is involved in the regulation of nitric oxide synthase (NOS) by metabolizing the free endogenous arginine derivatives N(omega)-methyl-L-arginine (MMA) and N(omega),N(omega)-dimethyl-L-arginine (ADMA), which are competitive inhibitors of NOS. Here, we present high-resolution crystal structures of DDAH isoform 1 (DDAH-1) isolated from bovine brain in complex with different inhibitors, including S-nitroso-L-homocysteine and Zn2+, a regulator of this mammalian enzyme. The structure of DDAH-1 consists of a propeller-like fold similar to other arginine-modifying enzymes and a flexible loop, which adopts different conformations and acts as a lid at the entrance of the active site. The orientation and interaction mode of inhibitors in the active site give insight into the regulation and the molecular mechanism of the enzyme. The presented structures provide a basis for the structure-based development of specific DDAH-1 inhibitors that might be useful in the therapeutic treatment of NOS dysfunction-related diseases.

The endogenous nitric oxide synthase inhibitors L-N -methylarginine and L-N ,N -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.

The cysteine-hydrolase dimethylargininase-1 (DDAH-1) is an important regulator of NO production in mammalian tissue for which the availability of an inhibitor for clinics and research would be most appreciated. While studying the effect of the endogenously occurring S-nitroso-l-homocysteine on DDAH-1, an unusual N-thiosulfoximide modification was identified in the active site of the enzyme. Thus, S-nitroso-l-homocysteine in combination with the mechanism proposed herein offers a basis for the rational design of DDAH inhibitors.

Human metallothionein-3 (Zn(7)MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain, where it plays an important role in the homeostasis of the essential metal ions Cu(+) and Zn(2+). Like other mammalian metallothioneins (MT-1 and -2), the protein contains a M(II)(3)(CysS)(9) and a M(II)(4)(CysS)(11) cluster localized in two independent protein domains linked by a flexible hinge region. However, there is a substantially increased number of acidic residues in MT-3 (11 residues) compared with MT-2 (four residues) which may act as binding ligands for additional metal ions. In this study, the binding of Zn(2+), Ca(2+), and Mg(2+) to human Zn(7)MT-3 and its mutant lacking an acidic hexapeptide insert, Zn(7)MT-3(Delta55-60), was investigated and compared with the binding of Zn(7)MT-2. By using spectroscopic and spectrometric techniques, we demonstrate that one additional Zn(2+) binds with an apparent binding constant (K(app)) of approximately 100 microM to Zn(7)MT-3 and Zn(7)MT-3(Delta55-60), but not to Zn(7)MT-2. The changes in spectroscopic features of metal-thiolate clusters and gel filtration behavior reveal that the formation of Zn(8)MT-3 is immediate and is accompanied by a decrease in the Stokes radius (R(s)). The changes in the R(s) suggest a mutual approach of both protein domains. The fast binding of Zn(2+) is followed by a slow time-dependent protein dimerization. The binding of Zn(2+) to Zn(7)MT-3 is specific as in the presence of Ca(2+) and Mg(2+) only an alteration of the R(s) of Zn(7)MT-3 at substantially higher concentrations was observed. The significance of these findings for the biological role of MT-3 is discussed.

S-Transnitrosation is an important bioregulatory process whereby NO + equivalents are transferred between S-nitrosothiols and Cys of target proteins. This reaction proceeds through a common intermediate R-S-N(O À )-S-R9 and it has been proposed that products different from S-nitrosothiols may be formed in protein cavities. Recently, we have reported on the formation of such a product, an N-thiosulfoximide, at the active site of the Cys hydrolase dimethylargininase-1 (DDAH-1) upon reaction with S-nitroso-L-homocysteine (HcyNO). Here we have addressed the question of whether this novel product can also be formed with the endogenously occurring S-nitrosothiols S-nitroso-L-cysteine (CysNO) and S-nitrosoglutathione (GSNO). Further, to explore the reason responsible for the unique formation of an N-thiosulfoximide in DDAH-1 we have expanded these studies to cytidine triphosphate synthetase (CTPS), which shows a similar active site architecture. ESI-MS and activity measurements showed that the bulky GSNO does not react with both enzymes. In contrast, S-nitrosylation of the active site Cys occurred in DDAH-1 with CysNO and in CTPS with CysNO and HcyNO. Although kinetic analysis indicated that these compounds act as specific irreversible inhibitors, no N-thiosulfoximide was formed. The reasons likely responsible for the absence of the N-thiosulfoximide formation are discussed using molecular models of DDAH-1 and CTPS. In tissue extracts DDAH was inhibited only by HcyNO, with an IC 50 value similar to that of the isolated protein. Biological implications of these studies for the function of both enzymes are discussed.Keywords: CTP synthetase; dimethylargininase; glutamine amidotransferase; hyperhomocyst(e)inemia; S-nitrosothiols; nitric oxide; S-transnitrosation Abbreviations: ADI, arginine deiminase; ADMA, Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi