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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
Author(s) -
Shen Maryann L.,
Johnson Kenneth L.,
Mays Dennis C.,
Lipsky James J.,
Naylor Stephen
Publication year - 2000
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(20000530)14:10<918::aid-rcm966>3.0.co;2-p
Subject(s) - chemistry , aldehyde dehydrogenase , chromatography , electrospray ionization , adduct , alcohol dehydrogenase , proteolytic enzymes , metabolite , mass spectrometry , sulfoxide , peptide , enzyme , biochemistry , organic chemistry
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 µM. The inhibition of rlmALDH was found to be accompanied by a concomitant increase of ∼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. Copyright © 2000 John Wiley & Sons, Ltd.