TAILIEUCHUNG - Báo cáo khoa học: Reduction of S-nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry

Human alcohol dehydrogenase 3/glutathione-dependent formaldehyde dehydrogenase was shown to rapidly and irreversibly catalyse the reductive breakdown of S-nitroso-glutathione. The steady-state kinetics of S-nitrosogluta-thione reduction was studied for the wild-type and two mutated forms of human alcohol dehydrogenase 3, muta-tions that have previously been shown to affect the oxidative efficiency for the substrate S-hydroxymethylglutathione. | Eur. J. Biochem. 270 1249-1256 2003 FEBS 2003 doi Reduction of 5-nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry Jesper J. Hedberg William J. Griffiths Stina J. F. Nilssont and Jan-Olov Hoog From the Department of Medical Biochemistry and Biophysics Karolinska Institutet Stockholm Sweden Human alcohol dehydrogenase 3 glutathione-dependent formaldehyde dehydrogenase was shown to rapidly and irreversibly catalyse the reductive breakdown of S-nitroso-glutathione. The steady-state kinetics of S-nitrosogluta-thione 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-hydroxy-methylglutathione oxidation resulting in the highest catalytic efficiency yet identified for a human alcohol dehydrogenase. In a similar manner as for S-hydroxy-methylglutathione 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

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