Nitric oxide inhibits ornithine decarboxylase via S-nitrosylation of cysteine 360 in the active site of the enzyme

Ornithine decarboxylase is the initial and rate-limiting enzyme in the poly amine biosynthetic pathway. Polyamines are found in all mammalian cells and are required for cell growth. We previously demonstrated that N-hydroxyarg inine and nitric oxide inhibit tumor cell proliferation by inhibiting argin ase and ornithine decarboxylase, respectively, and, therefore, polyamine sy nthesis. In addition, we showed that nitric oxide inhibits purified ornithi ne decarboxylase by S-nitrosylation. Herein we provide evidence for the che mical mechanism by which nitric oxide and S-nitrosothiols react with cystei ne residues in ornithine decarboxylase to form an S-nitrosothiol(s) on the protein. The diazeniumdiolate nitric oxide donor agent 1-diethyl-2-hydroxy- 2-nitroso-hydrazine acts through an oxygen-dependent mechanism leading to f ormation of the nitrosating agents N2O3 and/or N2O4. S-Nitrosoglutathione i nhibits ornithine decarboxylase by an oxygen-independent mechanism likely b y S-transnitrosation. In addition, we provide evidence for the S-nitrosylat ion of 4 cysteine residues per ornithine decarboxylase monomer including cy steine 360, which is critical for enzyme activity. Finally S-nitrosylated o rnithine decarboxylase was isolated from intact cells treated with nitric o xide, suggesting that nitric oxide may regulate ornithine decarboxylase act ivity by S-nitrosylation in vivo.