Studies in vitro on the inactivation of mitochondrial rat-liver aldehyde dehydrogenase by the alcohol-sensitizing compounds cyanamide, 1-aminocyclopropanol and disulfiram.
The inhibition of the low-Km, rat-liver mitochondrial aldehyde dehydrogenase (ALDH) by the alcohol-sensitizing agents cyanamide, 1-aminocyclopropanol (ACP) and disulfiram was studied in vitro. All three compounds caused a progressive decline in the enzyme activity. Restoration of activity could not be achieved by gel-filtration, dilution or by the addition of excess thiol. High concentrations of acetaldehyde partly restored the activity of the cyanamide-inactivated enzyme but had no effects on the disulfiram- or ACP-inactivated enzyme. In the presence of saturating concentrations of the coenzyme (NAD+), the inactivation process followed first-order kinetics at fixed concentrations of the inhibitors. Plots of the apparent first-order rate constants against inhibitor concentration were curved, suggesting the formation of saturable, reversible holoenzyme-inhibitor complexes prior to the covalent reactions. In the absence of NAD+, the rate of inactivation by disulfiram was biphasic and considerably higher than that in the presence of NAD+. In contrast, no inactivation was obtained with cyanamide in the absence of NAD+. Likewise, the presence of NAD+ greatly promoted the inactivation by ACP. The esterase activity of the enzyme was also affected by the inhibitors, although to a lesser extent than was the dehydrogenase activity. The results obtained suggest that all three inhibitors inactivate the enzyme through covalent reactions with the thiol groups at the active site. It is proposed that binding of NAD+ limits access of disulfiram to the thiols at the active site but provides a situation that favours an electrophilic attack of cyanamide and ACP on the thiol groups.
D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors*; D000818:Animals; D003484:Cyanamide / Q000494:pharmacology*; D003486:Cyanides / Q000494:pharmacology*; D003521:Cyclopropanes / Q000494:pharmacology*; D004221:Disulfiram / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D051381:Rats
D003486:Cyanides; D003521:Cyclopropanes; C014318:1-aminocyclopropanol; D009243:NAD; D003484:Cyanamide; D000445:Aldehyde Oxidoreductases; D004221:Disulfiram