ENZYMOLOGY/MOLECULAR BIOLOGY OF ALDEHYDE DEHYDROGENASE

乙醛脱氢酶(ALDH)参与氧化 乙醇衍生的乙醛及其解毒作用 其他外源生物的种类。尽管纯粹的线粒体酶有 多年来一直被调查，对此知之甚少 活动站点。化学修饰研究和天然氨基 允许对预测中的酸替代或在 活动站点。最近获得了大鼠的编码基因 可以进行肝酶定点突变实验 测定活性部位的残留量。这件事会做到的 与大鼠和酵母线粒体ALDHs进行比较。酵母醛 脱氢酶将被提纯，并产生一个ALDH突变体。这个 突变体将作为细胞插入改变的酵母或 大鼠肝脏ALDHs在质粒上的表达。突变的细胞不会继续生长 乙醇可以通过表达添加的ALDH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 活动站点的候选者。辅酶的结合域 结合将通过在体外生产截短蛋白来研究。 从工程cDNA制备mNRA后的合成。一种能力 这些酶的形式与NAD亲和层析结合 柱将用于评估对NAD(H)结合的损害 域。新近发现的N-末端乙酰化的作用 将探索哺乳动物线粒体ALDHs。这将包括 它在允许新合成的酶与 体外翻译后的四聚体状态及其稳定性 它提供成熟的酶。整个项目被设计为 能够理解这种负责乙醛的酶 新陈代谢。更多地了解涉及的酶可能有助于 合理设计药物来抑制这种酶，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.