Identification of the cysteine residue in the active site of horse liver mitochondrial aldehyde dehydrogenase.
Aldehyde dehydrogenase catalyzes the oxidation of aldehydes to acids through the formation of a covalent intermediate. It has been postulated that a cysteine residue could be acting as the active site nucleophilic group. Although N-ethylmaleimide was found to react with many cysteines it was possible by doing the reaction in the presence of chloral hydrate, a substrate analog which functions as a competitive inhibitor, to label cysteine at position 49 in the horse liver mitochondrial enzyme. The dehydrogenase activity was lost as the residue was modified, consistent with the possibility that the residue was an integral component of the active site of the enzyme. Cysteines at positions 162 and 369 also could be modified. It is suggested that cysteine 162 may function as part of a site capable of hydrolyzing nitrophenyl acetate. Details of the second site will appear in the accompanying paper (Tu, G. C., and Weiner, H. (1988) J. Biol. Chem. 263, 1218-1222). It appeared that the substrate-binding domain was in the N-terminal portion of the enzyme while the coenzyme binding domain was in the C-terminal portion. During this investigation 133 of the 500 residues of the horse liver enzyme were sequenced. These showed about 95% sequence identity with those of the human enzyme. Inasmuch as both beef and rat liver enzymes also share 95% identity with the human enzyme it can be expected that the results found with the horse liver enzyme can be applicable to all mammalian aldehyde dehydrogenase.
Tu
G C
GC
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Weiner
H
H
D000444:Aldehyde Dehydrogenase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002697:Chloral Hydrate / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006736:Horses; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping
D002697:Chloral Hydrate; D000444:Aldehyde Dehydrogenase; D003545:Cysteine; D005033:Ethylmaleimide