title: Reduction of S-nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry.
abstract: Human alcohol dehydrogenase 3/glutathione-dependent formaldehyde dehydrogenase was shown to rapidly and irreversibly catalyse the reductive breakdown of S-nitrosoglutathione. The steady-state kinetics of S-nitrosoglutathione reduction was studied for the wild-type and two mutated forms of human alcohol dehydrogenase 3, mutations that have previously been shown to affect the oxidative efficiency for the substrate S-hydroxymethylglutathione. Wild-type enzyme readily reduces S-nitrosoglutathione with a kcat/Km approximately twice the kcat/Km for S-hydroxymethylglutathione oxidation, resulting in the highest catalytic efficiency yet identified for a human alcohol dehydrogenase. In a similar manner as for S-hydroxymethylglutathione oxidation, the catalytic efficiency of S-nitrosoglutathione reduction was significantly decreased by replacement of Arg115 by Ser or Lys, supporting similar substrate binding. NADH was by far a better coenzyme than NADPH, something that previously has been suggested to prevent reductive reactions catalysed by alcohol dehydrogenases through the low cytolsolic NADH/NAD+ ratio. However, the major products of S-nitrosoglutathione reduction were identified by electrospray tandem mass spectrometry as glutathione sulfinamide and oxidized glutathione neither of which, in their purified form, served as substrate or inhibitor for the enzyme. Hence, the reaction products are not substrates for alcohol dehydrogenase 3 and the overall reaction is therefore irreversible. We propose that alcohol dehydrogenase 3 catalysed S-nitrosoglutathione reduction is of physiological relevance in the metabolism of NO in humans.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Jesper J
authors.initials: JJ
authors.lastname: Hedberg
authors.forename: William J
authors.initials: WJ
authors.lastname: Griffiths
authors.forename: Stina J F
authors.initials: SJ
authors.lastname: Nilsson
authors.forename: Jan-Olov
authors.initials: JO
authors.lastname: Höög
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D026422:S-Nitrosoglutathione / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D009243:NAD; D009569:Nitric Oxide; D009249:NADP; D026422:S-Nitrosoglutathione; C020145:formaldehyde dehydrogenase (glutathione); D000445:Aldehyde Oxidoreductases; D005978:Glutathione