INACTIVATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE BY A REACTIVE METABOLITE OF ACETAMINOPHEN AND MASS-SPECTRAL CHARACTERIZATION OF ANARYLATED ACTIVE-SITE PEPTIDE

Acetaminophen (4'-hydroxyacetanilide, APAP) is a widely used analgesic and antipyretic drug that can cause hepatic necrosis under some circu mstances via cytochrome P450-mediated oxidation to a reactive metaboli te, M-acetyl-p-benzoquinone imine (NAPQI). Although the mechanism of h epatocellular injury caused by APAP is not fully understood, it is kno wn that NAPQI forms covalent adducts with several hepatocellular prote ins. Reported here is the identification of one of these proteins as g lyceraldehyde-3-phosphate dehydrogenase [GAPDH, D-glyceraldehyde-3-pho sphate:NAD(+) oxidoreductase (phosphorylating), EC 1.2.1.12]. Two hour s after the administration of hepatotoxic doses of [C-14]APAP to mice, at a time prior to overt cell damage, hepatocellular GAPDH activity w as significantly decreased concurrent with the formation of a C-14-lab eled GAPDH adduct. A nonhepatotoxic regioisomer of APAP, 3'-hydroxyace tanilide (AMAP), was found to decrease GAPDH activity to a lesser exte nt than APAP, and radiolabel from [C-14]AMAP bound to a lesser extent to GAPDH at a time when its overall binding to hepatocellular proteins was almost equivalent to that of APAP. In order to determine the natu re of the covalent adduct between GAPDH and APAP, its major reactive a nd toxic metabolite, NAPQI, was incubated with purified porcine muscle GAPDH. Microsequencing analysis and fast atom bombardment mass spectr ometry (FAB-MS) with collision-induced dissociation (CID) were used to characterize one of the adducts as APAP bound to the cysteinyl sulfhy dryl group of Cys-149 in the active site peptide of GAPDH.