Modification of aldehyde dehydrogenase with dicyclohexylcarbodiimide: separation of dehydrogenase from esterase activity.
Dehydrogenase activity of the cytoplasmic (E1) isozyme of human liver aldehyde dehydrogenase (EC 1.2.1.3) was almost totally abolished (3% activity remaining) by preincubation with dicyclohexylcarbodiimide (DCC), while esterase activity with p-nitrophenyl acetate as substrate remained intact. The esterase reaction of the modified enzyme exhibited a hysteretic burst prior to achieving steady-state velocity; addition of NAD+ abolished the burst. The Km for p-nitrophenyl acetate was increased, but physicochemical properties remained unchanged. The selective inactivation of dehydrogenase activity was the result of covalent bond formation. Protection by NAD+ and chloral, saturation kinetics, and the stoichiometry and specificity of interaction indicated that the reaction of DCC occurred at the active site of the E1 isozyme. The results suggested the some amino acid other than aspartate or glutamate, possibly a cysteine residue, located on a large tryptic peptide of the E1 enzyme, may have reacted with DCC.
Abriola
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DP
Center of Alcohol Studies, Rutgers University, Piscataway, New Jersey 08855-0969.
Pietruszko
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D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D000595:Amino Acid Sequence; D002697:Chloral Hydrate / Q000031:analogs & derivatives / Q000494:pharmacology; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003956:Dialysis; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004950:Esterases / Q000737:chemistry / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000494:pharmacology; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism
D003432:Cross-Linking Reagents; D007527:Isoenzymes; D009243:NAD; D002697:Chloral Hydrate; C022010:glycine ethyl ester; D004024:Dicyclohexylcarbodiimide; D010088:Oxidoreductases; D000444:Aldehyde Dehydrogenase; D004950:Esterases; C021100:trichloroacetaldehyde; D005998:Glycine