Identification of the protein-drug adduct formed between aldehyde dehydrogenase and S-methyl-N,N-diethylthiocarbamoyl sulfoxide by on-line proteolytic digestion high performance liquid chromatography electrospray ionization mass spectrometry.
Disulfiram has been used clinically as an aversion therapy treatment for recovering alcoholics. One of its metabolites, S-methyl-N, N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), is currently believed by some to be the active metabolite in vivo. We demonstrate in this report that MeDTC-SO is a potent irreversible inhibitor of recombinant rat liver mitochondrial aldehyde dehydrogenase (rlmALDH), the enzyme responsible for oxidizing acetaldehyde formed during ethanol metabolism. Recombinant rlmALDH was inhibited by MeDTC-SO after in vitro incubation with an IC(50) = 4.62 microM. The inhibition of rlmALDH was found to be accompanied by a concomitant increase of approximately 100 Da to the molecular mass of the native enzyme as determined by on-line high performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (LC/MS), indicating that a covalent modification has occurred. To determine the site and structure of this covalent adduct, we developed a novel approach to characterize specific protein-drug interactions by linking a proteolytic enzyme digestion cartridge on-line with LC/MS. The on-line pepsin digestion LC/MS of MeDTC-SO-inhibited rlmALDH revealed an ion at MH(2)(2+) = 500.9, which was not present in the pepsin digestion of the non-inhibited enzyme. This peptide was tentatively attributed to the putative active site peptide (FNQGQC(301)C(302)C(303)) plus the adduct. This peptide was subjected to analysis by LC/MS/MS, which allowed us to determine that the covalent modification was associated with a single carbamoyl adduct at Cys-302, which has been shown to be the active site nucleophile of the enzyme.
Shen
M L
ML
Biomedical Mass Spectrometry and Functional Proteomics Facility Mayo Clinic/Foundation, Rochester, MN 55905, USA.
Johnson
K L
KL
Mays
D C
DC
Lipsky
J J
JJ
Naylor
S
S
D000427:Alcohol Deterrents / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D000444:Aldehyde Dehydrogenase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004050:Ditiocarb / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry / Q000379:methods*; D010434:Pepsin A; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D017421:Sequence Analysis
D000427:Alcohol Deterrents; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C073220:S-methyl N,N-diethylthiolcarbamate sulfoxide; D004050:Ditiocarb; D000444:Aldehyde Dehydrogenase; D010434:Pepsin A