MECHANISMS OF INHIBITION OF ALDEHYDE DEHYDROGENASE BY NITROXYL, THE ACTIVE METABOLITE OF THE ALCOHOL DETERRENT AGENT CYANAMIDE

Nitroxyl, produced in the bioactivation of the alcohol deterrent agent cyanamide, is a potent inhibitor of aldehyde dehydrogenase (AlDH); ho wever, the mechanism of inhibition of AlDH by nitroxyl has not been de scribed previously. Nitroxyl is also generated from Angeli's salt (Na2 N2O3) at physiological pH, and, indeed, Angeli's salt inhibited yeast AlDH in a time- and concentration-dependent manner, with IC50 values u nder anaerobic conditions with and without NAD(+) of 1.3 and 1.8 mu M, respectively. Benzaldehyde, a substrate for AlDH, competitively block ed the inhibition of this enzyme by nitroxyl in the presence of NAD(+) , but not in its absence, in accord with the ordered mechanism of this reaction. The sulfhydryl reagents dithiothreitol (5 mM) and reduced g lutathione (10 mM) completely blocked the inhibition of AlDH by Angeli 's salt. These thiols were also able:to partially restore activity to the nitroxyl-inhibited enzyme, the extent of reactivation being depend ent on the pH at which the inactivation occurred. This pH dependency i ndicates the formation of two inhibited forms of the enzyme, with an i rreversible form predominant at pH 7.5 and below, and a reversible for m predominant at pH 8.5 and above. The reversible form of the inhibite d enzyme is postulated to be an intra-subunit disulfide, while the irr eversible form is postulated to be a sulfinamide. Both forms of the in hibited enzyme are derived via a common N-hydroxysulfenamide intermedi ate produced by the addition of nitroxyl to active site cysteine thiol (s). BIOCHEM PHARMACOL 55;12:2007-2015, 1998. (C) 1998 Elsevier Scienc e Inc.