Aldehyde dehydrogenasee (ALDH) is involved in the oxidation of acetaldehyde derived from ethanol as well as in the detoxication of other xenobiotics. Though the pure mitochondrial enzyme has been investigated for a number of years little is known about the active site. Chemical modification studies and a natural amino acid substitution allowed for the prediction or residues ln the active site. Having recently obtained the cDNA coding for the rat liver enzyme site-directed mutagenesis experiments can be performed to determine the residues in the active site. This will be done with the rat and yeast mitochondrial ALDHs. Yeast aldehyde dehydrogenase will be Purified and a ALDH- mutant created. The mutant will serve as a cell ln which to insert altered yeast or rat liver ALDHs on Plasmids. The mutant cells will not grow on ethanol so growth can be restored by the expression of added ALDH genes. The yeast ALDH gene will be sequenced; chemical modification studies will be done with the yeast enzyme to probe the candidate of the active site. The binding domain of coenzyme binding will be studied by producing truncated protein in in vitro synthesis after preparing mNRA from engineered cDNAs. The ability of these enzyme forms to bind to NAD-affinity chromatography columns will be used to assess damage to the NAD(H) binding domain. The role of the recently found N-terminal acetylation of mammalian mitochondrial ALDHs will be explored. This will include its role in allowing the newly synthesized enzYme to associate to the tetrameric state after in vitro translations and the stability it affords mature enzymes. The overall project is designed to be able to understand this enzyme responsible for acetaldehyde metabolism. Knowing more about the enzyme involved could aid in a rational design of drugs to inhibit the enzyme and thus help deter alcohol abuse.
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