Thiols mediate superoxide-dependent NADH modification of glyceraldehyde-3-phosphate dehydrogenase.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is covalently modified by NAD in the presence of nitric oxide (NO) and dithiothreitol. Replacement of NAD with NADH in the presence of SIN-1 (3-morpholinosydnonimine) and dithiothreitol increased modification 25-fold. We now demonstrate that in contrast to NO-mediated attachment of NAD, covalent attachment of NADH to GAPDH proceeds in the presence of low molecular weight thiols, independent of NO. Removal of oxygen and transition metal ions inhibited modification, consistent with a role for reactive oxygen species; inhibition by superoxide dismutase, stimulation by xanthine oxidase/hypoxanthine, and the lack of an effect of catalase supported the hypothesis that superoxide, generated from thiol oxidation, was involved. Electrospray mass spectrometry showed covalent linkage of the NADH molecule to GAPDH. Characterization of the product of phosphodiesterase cleavage demonstrated that linkage to GAPDH occurred through the nicotinamide of NADH. Lys-C digestion of GAPDH, followed by peptide isolation by high performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight analysis, and Edman sequencing, demonstrated that NADH attachment occurred at Cys-149, the active-site thiol. This thiol linkage was stable to HgCl2. Thus, linkage of GAPDH to NADH, in contrast to NAD, occurs in the presence of thiol, is independent of NO, and is mediated by superoxide.
Rivera-Nieves
J
J
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA. JR3u@virginia.edu
Thompson
W C
WC
Levine
R L
RL
Moss
J
J
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002374:Catalase / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002772:Cholera Toxin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry*; D008666:Metalloendopeptidases; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000201:enzymology; D009243:NAD / Q000737:chemistry*; D009569:Nitric Oxide / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000494:pharmacology*; D014969:Xanthine Oxidase / Q000378:metabolism
D002614:Chelating Agents; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D009243:NAD; D013481:Superoxides; D009599:Nitroprusside; D009569:Nitric Oxide; C002385:linsidomine; D002772:Cholera Toxin; D008981:Molsidomine; D002374:Catalase; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D001205:Ascorbic Acid; D004229:Dithiothreitol