ENZYMOLOGY/MOLECULAR BIOLOGY OF ALDEHYDE DEHYDROGENASE

乙醛脱氢酶（ALDH）参与了 乙醇衍生的乙醛以及在解毒中 其他异生物质。 虽然纯线粒体酶 经过多年的研究，人们对它知之甚少。 活性部位 化学修饰研究和天然氨基 酸取代允许的预测或残基在 活性部位 最近获得了编码大鼠 可以进行肝酶定点诱变实验 以确定活性位点中的残基。 为此将 与大鼠和酵母线粒体ALDHs进行比较。 酵母醛 将纯化脱氢酶并产生ALDH突变体。 的 突变体将作为一个细胞，在其中插入改变的酵母或 大鼠肝ALDH在血浆中的表达。 变异细胞不会在 因此，通过表达添加的ALDH可以恢复生长 基因. 酵母ALDH基因将被测序;化学 将用酵母酶进行修饰研究， 活性位点的候选者。 辅酶的结合结构域 结合将通过在体外产生截短的蛋白质来研究 在从工程化cDNA制备mNRA之后进行合成。 的能力 这些酶形式结合NAD亲和层析 柱将用于评估NAD（H）结合的损害 域 最近发现的N-末端乙酰化的作用， 将探索哺乳动物线粒体ALDH。 这将包括 它的作用是使新合成的酶与 体外翻译后的四聚体状态和稳定性 它提供成熟的酶。 整个项目旨在 能够理解这种负责乙醛的酶 新陈代谢. 更多地了解相关的酶可以帮助 合理设计药物来抑制这种酶， 制止酗酒。

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.