ACTIVE SITE OF NAD(H) DEPENDENT ENZYMES

NAD(H)是许多酶的辅酶。 除了它在氧化还原中的作用之外 反应中，NAD(H)还参与代谢的许多方面 直到最近才被承认的法规。一个 了解这些酶的结构是很重要的。 以了解与此相关的生化过程 用这些酶。然而，在所有依赖NAD(H)的人中 酶，只有结构上的几个简单的氧化还原 已经对酶进行了研究。 在本应用程序中，我们建议研究两个 NAD(H)依赖酶，牛心脏线粒体NADH 脱氢酶和NADH-NAD+转氢酶亲和 贴标签技术。这两种酶都很重要 参与细胞能量产生过程的酶。这个 线粒体NAD(H)脱氢酶也被证明可以 部分参与了心肌氧中毒的机制，1- 甲基-4-苯基-1，2，3，6-四氢吡啶的毒性 与帕金森氏症有关。尽管许多研究人员已经 对这些酶表现出极大的兴趣，没有结构信息 对这两种酶都是可用的，因为 这些酶制剂。在这个拟议的项目中，活跃的 这些酶的位置将通过标记进行鉴定 六种NAD+亲和探针的放射性衍生物。这个 经这些探针修饰的多肽将被表征为 微测序、氨基酸序列分析和快原子 轰击质谱仪。其氨基酸序列 三种酶的修饰多肽将与那些 在表征良好的NAD(H)-的活性中心区域- 依赖的酶。这项调查将确定重要的 这两种酶的区域。通过以下方式生成的信息 这一研究将对进一步理解分子生物学有价值。 这些酶催化的反应的机理。 此外，这些结构信息将非常有助于 未来这两种酶的结构和功能研究 分子克隆和定点突变实验。

NAD(H) functions as the coenzyme of a large number of enzymes. In addition to its well documented roles in oxidation-reduction reactions, NAD(H) is also involved in many aspects of metabolic regulation which have only recently been recognized. An understanding of the structure of these enzymes will be important for the understanding of those biochemical processes associated with these enzymes. However, among all NAD(H) dependent enzymes, only the structure of a few simple oxido-reduction enzymes have been investigated. In this application we propose to investigate the active site of two NAD(H) dependent enzymes, bovine heart mitochondrial NADH dehydrogenase, and NADH-NAD+ transhydrogenase by affinity labeling techniques. Both of these enzymes are important enzymes involved in the cellular energy production process. The mitochondrial NAD(H) dehydrogenase has also been shown to take part in mechanisms inducing cardiac oxygen toxicity and 1- methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity related to Parkinson Disease. Although many researchers have shown great interest in these enzymes, no structural information is yet available for either enzyme because of the heterogenity of these enzyme preparations. In this proposed project, the active sites of these enzymes will be identified by labeling with radioactive derivatives of six affinity probes of NAD+. The peptides modified by these probes will be characterized by microsequencing, amino acid sequences of analysis, and fast atom bombardment mass spectrometry. The amino acid sequences of the modified peptides of three enzymes will be compared to those at the active site regions of well characterized NAD(H)- dependent enzymes. This investigation will identify important regions of these two enzymes. The information generated through this study will be valuable for further understanding the molecular mechanism of the reactions these enzymes catalyze. Furthermore, this structural information will be very useful for future structure-function study of these two enzymes by molecular cloning and site specific mutagenesis experiments.