The effects of disulfiram and related compounds on equine hepatic alcohol dehydrogenase.
The reversible inhibition and the irreversible inactivation of equine hepatic alcohol dehydrogenase by disulfiram have been investigated. Disulfiram was found to be a potent competitive reversible inhibitor with KEO,I values at pH 7.0 and 10.0 of 50 microM and 30 microM, respectively. Reversible monodentate binding to the active site zinc is indicated by comparison with related compounds. Disulfiram was also found to chemically modify and inactivate the enzyme in an irreversible reaction, which proceeds via the formation of a reversible enzyme-disulfiram binary complex with a dissociation constant at pH 7.0 of 30 microM. The inactivation reaction has been studied over the pH 6.0 to 10.0 range. The dissociation constants for binding to the enzyme and the apparent first-order rate constants for inactivation have been determined as a function of pH. A pKa of 8.3 for the free enzyme has been assigned to the zinc-water ionization. Similar inhibition and affinity labelling kinetics are exhibited by diethyldithiocarbamate and by 2,2'- and 4,4'-dipyridyl disulphide, which have similar enzyme "on" velocity pKa values of 8.3 and 8.2, respectively. The enzyme is competitively protected from inactivation with disulfiram by 2,2'-dipyridyl, 1,7'-phenanthroline, acetone, and ethanol, all of which combine with the active site zinc to form binary complexes. Acetate gave mixed protection against inactivation due to an additional interaction with the anion binding site of the enzyme. In view of the effect of disulfiram on ethanol metabolism and the polyol pathway, its importance as an aversive drug are considered.
Langeland
B T
BT
Institute of Biochemistry, University of Oslo, Blindern, Norway.
McKinley-McKee
J S
JS
D000345:Affinity Labels / Q000494:pharmacology; D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D004221:Disulfiram / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000378:metabolism; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D010084:Oxidation-Reduction
D000345:Affinity Labels; D007093:Imidazoles; C000314:alpha-bromo-beta-(5-imidazolyl)propionic acid; D000431:Ethanol; D000426:Alcohol Dehydrogenase; D004221:Disulfiram