ENZYMOLOGY/MOLECULAR BIOLOGY OF ALDEHYDE DEHYDROGENASE

乙醛脱氢酶 (ALDH) 参与氧化 乙醇衍生的乙醛以及解毒作用 其他外源物质。 尽管纯线粒体酶具有 经过多年的调查，人们对此知之甚少 活跃站点。 化学修饰研究和天然氨基 酸取代允许预测或残基 活跃站点。 最近获得了大鼠的 cDNA 编码 可以进行肝酶定点突变实验 以确定活性位点的残基。 这将完成 与大鼠和酵母线粒体 ALDH 一起使用。 酵母醛 脱氢酶将被纯化并产生 ALDH 突变体。 的 突变体将作为细胞插入改变的酵母或 质粒上的大鼠肝脏 ALDH。 突变细胞将无法生长 乙醇，因此可以通过添加 ALDH 的表达来恢复生长 基因。 酵母 ALDH 基因将被测序；化学 将用酵母酶进行修饰研究以探测 活性位点的候选者。 辅酶的结合域 将通过在体外产生截短的蛋白质来研究结合 从工程化的 cDNA 制备 mNRA 后进行合成。 能力 这些酶形式与 NAD 亲和层析结合 色谱柱将用于评估 NAD(H) 结合的损伤 域。 最近发现的N端乙酰化的作用 将探索哺乳动物线粒体 ALDH。 这将包括 它在允许新合成的酶结合到 体外翻译后的四聚体状态及其稳定性 它提供成熟的酶。 整个项目的设计目的是 能够理解这种负责乙醛的酶 新陈代谢。 了解更多有关所涉及酶的信息可能有助于 合理设计药物来抑制酶，从而帮助 阻止酗酒。

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.