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 dithi othreitol increased modification 25-fold. We now demonstrate that in contra st to NO-mediated attachment of NAD, covalent attachment of NADH to GAPDH p roceeds in the presence of low molecular weight thiols, independent of NO. Removal of oxygen and transition metal ions inhibited modification, consist ent with a role for reactive oxygen species; inhibition by superoxide dismu tase, stimulation by xanthine oxidase/hypoxanthine, and the lack of an effe ct of catalase supported the hypothesis that superoxide, generated hom thio l oxidation, was involved. Electrospray mass spectrometry showed covalent L inkage of the NADH molecule to GAPDH Characterization of the product of pho sphodiesterase cleavage demonstrated that linkage to GAPDH occurred through the nicotinamide of NADH. Lys-C digestion of GAPDH, followed by peptide is olation by high performance liquid chromatography, matrix-assisted laser de sorption ionization time-of-flight analysis, and Ed-man sequencing, demonst rated 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 cont rast to NAD, occurs in the presence of thiol, is independent of NO, and is mediated by superoxide.