Posttranslational modification of glyceraldehyde-3-phosphate dehydrogenase by S-nitrosylation and subsequent NADH attachment.
Nitric oxide (NO)-related activity has been associated with an NAD+-dependent modification of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, the mechanism by which NO effects covalent attachment of nucleotide and its role in regulation of enzyme activity are controversial. Recent studies have shown that S-nitrosylation of GAPDH (Cys149) initiates subsequent modification by the pyridinium cofactor. Here we show that NADH rather than NAD+ is the preferred substrate. Transnitrosation from active site S-nitrosothiol to the reduced nicotinamide ring system appears to facilitate protein thiolate attack on the enzyme-bound cofactor. This results in attachment of the intact NADH molecule. Moreover, we find that S-nitrosylation of GAPDH is responsible for reversible enzyme inhibition, whereas attachment of NADH accounts for irreversible enzyme inactivation. S-Nitrosylation may serve to protect GAPDH from oxidant inactivation in settings of cytokine overproduction and to regulate glycolysis. NADH attachment is more likely to be a pathophysiological event associated with inhibition of gluconeogenesis.
Mohr
S
S
Faculty of Biology, University of Konstanz, P.O. Box 5560 M612, 78434 Konstanz, Germany.
Stamler
J S
JS
Brüne
B
B
D000818:Animals; D002250:Carbon Radioisotopes; D003545:Cysteine; D005943:Gluconeogenesis; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000378:metabolism*; D006019:Glycolysis; D006706:Homeostasis; D006801:Humans; D007231:Infant, Newborn; D018482:Muscle, Skeletal / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011865:Radioisotope Dilution Technique
D002250:Carbon Radioisotopes; D010761:Phosphorus Radioisotopes; D009243:NAD; D009569:Nitric Oxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine