STRUCTURAL STUDIES OF NUCLEOTIDE DEPENDENT ENZYMES

核苷酸在几乎所有的生物化学过程中起着重要的作用 它们为DNA和RNA提供构建模块，充当货币 能量在各种代谢途径，作为调节os酶 活性，并作为瞬时电子受体和供体。 的 本研究计划的目的是了解在结构层面上， 核苷酸在三种酶的整体酶机制中的作用 特异性蛋白质系统：乙酰辅酶A羧化酶，即Mg 2 +-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+进行 活动 对于所提议的研究，结合定点诱变 实验，X射线晶体学和动力学测量将是 就业。 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， 很容易分离出拟定研究所需的蛋白质量 来自大肠杆菌中的过表达系统，以及适合于 获得了X射线分析。 此外，这些蛋白质发挥 重要的代谢作用，与健康相关的问题， 下面讨论。 乙酰辅酶A羧化酶催化的第一个关键步骤， 通过将乙酰辅酶A转化为丙二酰辅酶A来生物合成长链脂肪酸 检验报告。 丙二酰辅酶A的产生也是一种速率控制 这条生化途径的反应。 脂肪酸合成受损 会导致严重的心血管问题 因此 对乙酰辅酶A羧化酶的研究对于 了解正常的脂肪酸代谢。 那霉素 核苷酸转移酶催化卡那霉素的失活， 一种氨基糖苷类抗生素，用于治疗 需氧革兰氏阴性菌。 本文提出的结构研究 资助申请将提供有关 这种酶的活性位点，并可能最终导致设计 氨基糖苷类抗生素对酶的攻击有抵抗力。 最后，UDP-半乳糖4-差向异构酶，Leloir途径中的第三种酶， 将半乳糖转化为葡萄糖，在适当的半乳糖中起着重要作用。 新陈代谢. 在这个代谢循环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.