STRUCTURAL STUDIES OF NUCLEOTIDE DEPENDENT ENZYMES

核苷酸在几乎所有的生化过程中都扮演着重要的角色。 它们为DNA和RNA提供构建块，充当 能量存在于各种代谢途径中，是一种可调节的os酶。 活性，并作为瞬时电子受体和供体。这个 本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作用机制中的作用 特定蛋白质系统：乙酰辅酶A羧基酶，即镁离子-三磷酸腺苷 依赖的，卡那霉素核苷酸转移酶，可以利用ATP，GTP，或 UTP为底物，需要NAD+的UDP-半乳糖4-差向异构酶 活动。 在拟议的研究中，结合了定点突变 实验、x射线结晶学和动力学测量 受雇的。之所以选择这些蛋白质系统进行研究，是因为它们具有 已经被生化和生物物理技术很好地描述了， 建议的研究所需的蛋白质数量很容易分离出来 在大肠杆菌中的过度表达系统，以及适合于 获得了X射线分析结果。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，如 下面将进行讨论。 乙酰辅酶A羧基酶催化的第一步是 乙酰辅酶A转化为丙二酸基生物合成长链脂肪酸 可口可乐。丙二酰辅酶A的生产也是速率控制之一 这一生化途径的反应。脂肪酸合成障碍 会导致严重的心血管问题。因此， 提出的乙酰辅酶A羧化酶的研究对于 了解正常的脂肪酸代谢。卡那霉素 核苷酰转移酶催化卡那霉素的失活 氨基糖苷类抗生素在治疗慢性支气管炎感染中的应用 需氧革兰氏阴性细菌。在本报告中提出的结构研究 奖助金申请将提供有关 这种酶的活性部位，并可能最终导致设计 能抵抗酶攻击的氨基糖苷类抗生素。 最后，Leloir途径中的第三种酶-UDP-半乳糖4-差向异构酶 它将半乳糖转化为葡萄糖，在正常的半乳糖中起着主要作用 新陈代谢。新陈代谢周期中的缺陷会导致各种 疾病状态，包括半乳糖血症。

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.