STRUCTURAL STUDIES OF NUCLEOTIDE DEPENDENT ENZYMES

核苷酸依赖性酶的结构研究

• 批准号: 2430216
• 负责人: Hazel M. Holden
• 金额: $ 20.12万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-06-01 至 1998-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2430216
• 关键词: X ray crystallography; acetyl coA carboxylase; adenosine triphosphate; enzyme mechanism; enzyme structure; enzyme substrate analog; isomerase; kanamycin; mutant; nucleotides; nucleotidyltransferase; protein purification; site directed mutagenesis; structural biology; uridine diphosphate

Nucleotides serve important roles in virtually all biochemical processes where they provide building blocks for DNA and RNA, act as currencies of energy in various metabolic pathways, serve as modulatory os enzyme activity, and function as transient electron acceptors and donors. The objective of this research program is to understand on a structural level the role of nucleotides in the overall enzymatic mechanisms of three specific protein systems: acetyl CoA carboxylase which is Mg2+-ATP dependent, kanamycin nucleotidyltransferase which can employ ATP, GTP, or UTP as a substrate, and UDP-galactose 4-epimerase which requires NAD+ for activity. For the proposed studies, a combination of site-directed mutagenesis experiments, x-ray crystallography, and kinetic measurements will be employed. These protein systems were chosen for study because they have been well characterized by both biochemical and biophysical techniques, the amount of protein necessary for the proposed studies is readily isolated from over-expression systems in Escherichia coli, and crystals suitable for x-ray analyses have been obtained. In addition, these proteins play important metabolic roles that are relevant to health-related issues as discussed below. Acetyl CoA carboxylase catalyzes the first committed step in the biosynthesis of long chain fatty acids by converting acetyl-CoA to malonyl- CoA. The production of malonyl-CoA is also one of the rate-controlling reactions for this biochemical pathway. Impairment of fatty acid synthesis can lead to significant cardio-vascular problems. Consequently, the proposed studies of acetyl CoA carboxylase are critically important for understanding normal fatty acid metabolism. Kanamycin nucleotidyltransferase catalyzes the inactivation of kanamycin, an aminoglycoside antibiotic used in the treatment of infections due to aerobic Gram-negative bacteria. The structural studies proposed in this grant application will provide three-dimensional information concerning the active site of this enzyme and may ultimately lead to the design of aminoglycoside antibiotics that are resistant to attack by the enzyme. Finally, UDP-galactose 4-epimerase, the third enzyme in the Leloir pathway which converts galactose to glucose, plays a major role in proper galactose metabolism. Deficiencies in this metabolic cycle can lead to various diseased states including galactosemia.

核苷酸在几乎所有生化过程中都发挥着重要作用 它们为 DNA 和 RNA 提供构建模块，充当货币 各种代谢途径中的能量，充当调节性酶 活性，并充当瞬态电子受体和供体。 这 该研究计划的目标是在结构层面上理解 核苷酸在三种酶的整体酶促机制中的作用 特定蛋白质系统：乙酰辅酶A羧化酶，即Mg2+-ATP 依赖的卡那霉素核苷酸转移酶，其可以利用 ATP、GTP 或 UTP 作为底物，UDP-半乳糖 4-差向异构酶需要 NAD+ 活动。 对于拟议的研究，结合了定点诱变 实验、X 射线晶体学和动力学测量将 受雇。 选择这些蛋白质系统进行研究是因为它们具有 通过生物化学和生物物理技术得到了很好的表征， 拟议研究所需的蛋白质量很容易分离 来自大肠杆菌的过度表达系统，以及适合的晶体 已获得X射线分析。 此外，这些蛋白质还发挥着 与健康相关问题相关的重要代谢作用，例如 下面讨论。 乙酰辅酶A羧化酶催化第一个关键步骤 通过将乙酰辅酶A转化为丙二酰-来生物合成长链脂肪酸 辅酶A。 丙二酰辅酶A的产生也是速率控制之一 该生化途径的反应。 脂肪酸合成受损 可能导致严重的心血管问题。 因此， 乙酰辅酶A羧化酶的拟议研究对于 了解正常的脂肪酸代谢。 卡那霉素 核苷酸转移酶催化卡那霉素失活，卡那霉素是一种 氨基糖苷类抗生素用于治疗感染 需氧革兰氏阴性菌。 本文提出的结构研究 拨款申请将提供有关以下方面的三维信息 该酶的活性位点，并可能最终导致设计 氨基糖苷类抗生素能够抵抗酶的攻击。 最后，UDP-半乳糖4-差向异构酶，Leloir 途径中的第三种酶 它将半乳糖转化为葡萄糖，在适当的半乳糖中起着重要作用 代谢。 这个代谢循环的缺陷会导致各种 疾病状态，包括半乳糖血症。