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Specific reactions of S-nitrosothiols with cysteine hydrolases: A comparative study between dimethylargininase-1 and CTP synthetase

Oliver Braun, Markus Knipp¹, Serge Chesnov², and Milan Vaák

Department of Biochemistry, University of Zürich, CH-8057 Zürich, Switzerland

(RECEIVED December 12, 2006; FINAL REVISION April 27, 2007; ACCEPTED April 27, 2007)

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^–)–S–R' 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 IC₅₀ 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

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