Inhibition of aldehyde dehydrogenase by disulfiram and its metabolite methyl diethylthiocarbamoyl-sulfoxide.
Disulfiram (DSF) is presently the only available drug used in the aversion therapy of recovering alcoholics. It acts by inhibiting aldehyde dehydrogenase (ALDH), leading to high blood levels of acetaldehyde. The in vitro inhibition of ALDH by DSF and its metabolites was systematically studied by combined enzyme inhibition assay with direct molecular weight determination of the same sample using electrospray ionization-mass spectrometry (ESI-MS). Enzyme activity was measured after incubating yeast ALDH (yALDH) with excess concentrations of DSF, methyl diethyldithiocarbamate (MeDDC) and methyl diethylthiocarbamoyl-sulfoxide (MeDTC-SO) and then subjected to analysis by ESI-MS. Addition of DSF resulted in complete enzyme inhibition; however, ESI-MS analysis demonstrated no discernible shift in molecular weight, indicating that no intermolecular adduct was formed with the protein. Treatment of yALDH with MeDTC-SO also completely abolished yALDH activity with a concomitant increase of + approximately 100 Da in the molecular mass of the enzyme. This indicated formation of a covalent carbamoyl protein adduct. Furthermore, the effects of dithiothreitol (DTT) were examined on samples of inhibited protein in vitro. At pH 7.5, DTT completely reversed inhibition after DSF treatment. yALDH inhibited by MeDTC-SO could not be recovered by DTT at pH 7.5, but at pH 9 the enzymic activity was fully restored and a mass loss of approximately 100 Da was noted. This observations are consistent with mechanisms where inhibition of yALDH by DSF in vitro involves oxidation of the active site, whereas MeDTC-SO forms a covalent adduct with the protein in vitro resulting in cessation of enzyme activity.
Veverka
K A
KA
Department of Pharmacology, Mayo Clinic Foundation, Rochester, MN 55905, USA.
Johnson
K L
KL
Mays
D C
DC
Lipsky
J J
JJ
Naylor
S
S
D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors*; D004221:Disulfiram / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D004050:Ditiocarb / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D013454:Sulfoxides / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D013454:Sulfoxides; C016571:methyl diethyldithiocarbamate; D004050:Ditiocarb; D000444:Aldehyde Dehydrogenase; D004229:Dithiothreitol; D004221:Disulfiram