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

Here we show that in extrahepatic methionine adenosyltransferase replacemen t of a single amino acid (glycine 120) by cysteine is sufficient to create a functional nitric oxide binding site without affecting the kinetic proper ties of the enzyme. When wild-type and mutant methionine adenosyltransferas e were incubated with S-nitrosoglutathione the activity of the wild-type re mained unchanged whereas the activity of the mutant enzyme decreased marked ly, The mutant enzyme was found to be S-nitrosylated upon incubation with t he nitric oxide donor. Treatment of the S-nitrosylated mutant enzyme with g lutathione removed most of the S-nitrosothiol groups and restored the activ ity to control values. In conclusion, our results suggest that functional S -nitrosylation sites can develop from existing structures without drastic o r large-scale amino acid replacements. (C) 1999 Federation of European Bioc hemical Societies.