title: Creation of a functional S-nitrosylation site in vitro by single point mutation.
abstract: 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.
authors.affiliation: Department of Medicine, University of Navarra, 31008, Pamplona, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Castro
authors.forename: F A
authors.initials: FA
authors.lastname: Ruiz
authors.forename: I
authors.initials: I
authors.lastname: Pérez-Mato
authors.forename: M M
authors.initials: MM
authors.lastname: Sánchez del Pino
authors.forename: L
authors.initials: L
authors.lastname: LeGros
authors.forename: A M
authors.initials: AM
authors.lastname: Geller
authors.forename: M
authors.initials: M
authors.lastname: Kotb
authors.forename: F J
authors.initials: FJ
authors.lastname: Corrales
authors.forename: J M
authors.initials: JM
authors.lastname: Mato
mesh_terms: D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008716:Methionine Adenosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D026422:S-Nitrosoglutathione
chemical_list: D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D011994:Recombinant Proteins; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D008716:Methionine Adenosyltransferase; D005978:Glutathione; D003545:Cysteine