Creation of a functional S-nitrosylation site in vitro by single point mutation

• Published in: FEBS letters 1999-10, Vol.459 (3), p.319-322
• Main Authors: Castro, Carmen, Ruiz, Félix A., Pérez-Mato, Isabel, Sánchez del Pino, Manuel M., LeGros, Leighton, Geller, Arthur M., Kotb, Malak, Corrales, Fernando J., M. Mato, José
• Format: Article
• Language: English
• Subjects: AdoMet, S-adenosylmethionine; CBP, calmodulin binding peptide; Cysteine - genetics; Cysteine - metabolism; Glutathione - analogs & derivatives; Glutathione - pharmacology; GSH, glutathione; GSNO, S-nitrosoglutathione; Humans; MAT, methionine adenosyltransferase; Methionine adenosyltransferase; Methionine Adenosyltransferase - antagonists & inhibitors; Methionine Adenosyltransferase - genetics; Methionine Adenosyltransferase - metabolism; Mutagenesis, Site-Directed; Nitric Oxide - metabolism; Nitric Oxide Donors - pharmacology; Nitroso Compounds - pharmacology; NO, nitric oxide; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - metabolism; S-Nitrosoglutathione; S-Nitrosylation; SNO, nitrosothiol
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(99)01267-3

Here we show that in extrahepatic methionine adenosyltransferase replacement of a single amino acid (glycine 120) by cysteine is sufficient to create a functional nitric oxide binding site without affecting the kinetic properties of the enzyme. When wild-type and mutant methionine adenosyltransferase were incubated with S-nitrosoglutathione the activity of the wild-type remained unchanged whereas the activity of the mutant enzyme decreased markedly. The mutant enzyme was found to be S-nitrosylated upon incubation with the nitric oxide donor. Treatment of the S-nitrosylated mutant enzyme with glutathione removed most of the S-nitrosothiol groups and restored the activity to control values. In conclusion, our results suggest that functional S-nitrosylation sites can develop from existing structures without drastic or large-scale amino acid replacements.