ACTIVE SITE OF NAD(H) DEPENDENT ENZYMES

NAD(H) 依赖性酶的活性位点

• 批准号: 3292597
• 负责人: Shiuan Chen
• 金额: $ 12.73万
• 依托单位: BECKMAN RES INST OF THE CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292597
• 关键词: affinity labeling; chemical structure function; cow; enzyme mechanism; enzyme structure; heart; high performance liquid chromatography; maleimides; mass spectrometry; mitochondria; nicotinamide adenine dinucleotide; protein engineering; protein sequence

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.

NAD（H）是许多酶的辅酶。 除了其在氧化还原中的作用外， NAD（H）还参与代谢的许多方面， 这是最近才得到承认的。 一个 了解这些酶的结构将是重要的 对于理解那些与生物化学过程相关的 这些酶。 然而，在所有NAD（H）依赖者中， 酶，只有几个简单的氧化还原结构 酶已被研究。 在本申请中，我们提出研究两个 NAD（H）依赖性酶，牛心脏线粒体NADH 脱氢酶和NADH-NAD+转氢酶 标签技术。 这两种酶都很重要 参与细胞能量生产过程的酶。 的 线粒体NAD（H）脱氢酶也被证明可以 参与诱导心脏氧毒性的机制，1- 甲基-4-苯基-1，2，3，6-四氢吡啶（MPTP）毒性 与帕金森病有关。 尽管许多研究人员 对这些酶很感兴趣，没有结构信息 由于酶的异质性， 这些酶制剂。 在这个项目中，积极的 这些酶的位点将通过用 NAD+的六种亲和探针的放射性衍生物。 的 由这些探针修饰的肽的特征在于 微测序、氨基酸序列分析和快速原子分析 轰击质谱法 的氨基酸序列 将三种酶的修饰肽与那些 在充分表征的NAD（H）- 依赖酶 这次调查将确定重要的 这两种酶的区域。 通过以下方式生成的信息 本研究对进一步了解植物的分子生物学特性具有重要意义。 这些酶催化的反应机制。 此外，这些结构信息将非常有用， 这两种酶的未来结构-功能研究， 分子克隆和位点特异性诱变实验。