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Creation of a functional S ‐nitrosylation site in vitro by single point mutation
Author(s) -
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
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01267-3
Subject(s) - methionine adenosyltransferase , mutant , cysteine , s nitrosylation , methionine , biochemistry , nitrosylation , enzyme , nitric oxide , chemistry , glycine , amino acid , s nitrosoglutathione , enzyme assay , wild type , mutation , in vitro , glutathione , organic chemistry , gene
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.