Abstract
Ornithine decarboxylase is the initial and rate-limiting enzyme in the polyamine biosynthetic pathway. Polyamines are found in all mammalian cells and are required for cell growth. We previously demonstrated that N-hydroxyarginine and nitric oxide inhibit tumor cell proliferation by inhibiting arginase and ornithine decarboxylase, respectively, and, therefore, polyamine synthesis. In addition, we showed that nitric oxide inhibits purified ornithine decarboxylase by S-nitrosylation. Herein we provide evidence for the chemical mechanism by which nitric oxide and S-nitrosothiols react with cysteine 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 formation of the nitrosating agents N(2)O(3) and/or N(2)O(4). S-Nitrosoglutathione inhibits ornithine decarboxylase by an oxygen-independent mechanism likely by S-transnitrosation. In addition, we provide evidence for the S-nitrosylation of 4 cysteine residues per ornithine decarboxylase monomer including cysteine 360, which is critical for enzyme activity. Finally S-nitrosylated ornithine decarboxylase was isolated from intact cells treated with nitric oxide, suggesting that nitric oxide may regulate ornithine decarboxylase activity by S-nitrosylation in vivo.
Highlights
Ornithine decarboxylase (ODC),1 the rate-limiting enzyme in putrescine synthesis, catalyzes the conversion of ornithine to putrescine and is essential for polyamine synthesis in mammalian cells
We previously demonstrated that N-hydroxyarginine and nitric oxide inhibit tumor cell proliferation by inhibiting arginase and ornithine decarboxylase, respectively, and, polyamine synthesis
We set forth the hypothesis that GSNO acts by participating in an S-transnitrosation reaction with cysteine residue(s) on ODC, whereas DEA/NO likely acts through the reaction of NO with O2 to form N2O3 and N2O4, both of which are nitrosating agents (Fig. 2)
Summary
Ornithine decarboxylase (ODC), the rate-limiting enzyme in putrescine synthesis, catalyzes the conversion of ornithine to putrescine and is essential for polyamine synthesis in mammalian cells. Many early studies indicate that polyamine synthesis is enhanced during growth and that growthpromoting stimuli lead to increases in polyamine biosynthesis Direct evidence for this has been provided by experiments in which polyamine synthesis was prevented in mammalian cells in culture by mutations to key enzymes (such as ODC) or by the application of enzyme inhibitors (2, 4 – 6). We have consistently observed cyclic GMP-independent cytostatic effects of NO in a variety of mammalian cell types including Caco-2 human tumor cells, murine macrophages, and rat aortic endothelial and smooth muscle cells [15, 16].2 One such mechanism appears to be the inhibition of ODC by NO. The objective of this study was 3-fold: 1) to provide further evidence for the S-nitrosylation of ODC and the chemical mechanism by which it is S-nitrosylated, 2) to determine whether Cys-360, the critical cysteine residue in the active site of ODC, is S-nitrosylated, and 3) to determine whether ODC is S-nitrosylated in intact cells
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