MOLECULAR BASIS OF CELLULAR CONTROL MECHANISMS

细胞控制机制的分子基础

• 批准号: 2174665
• 负责人: EVAN R KANTROWITZ
• 金额: $ 20.84万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2174665
• 关键词: DNA replication; X ray crystallography; active sites; allosteric site; aspartate carbamoyltransferase; chemical kinetics; chemical stability; chemical substitution; circular dichroism; conformation; enzyme activity; enzyme induction /repression; enzyme mechanism; enzyme reconstitution; enzyme structure; gel electrophoresis; hybrid enzyme; intermolecular interaction; ion exchange chromatography; low angle X ray diffraction analysis; mutant; nuclear magnetic resonance spectroscopy; polymerase chain reaction; protein engineering; protein sequence; purine /pyrimidine metabolism; regulatory gene; site directed mutagenesis

The long term objectives of this project are to acquire a molecular level understanding of the mechanisms that govern cellular control of metabolic pathways. In particular, to study the actual protein molecules involved in the regulatory process, to examine the molecular basis by which an enzyme can regulate its own activity, and to determine how the cell can regulate the biosynthesis of that particular enzyme. Of all the metabolic pathways, the biosynthesis of the purines and pyrimidines, which are required in equal amounts for DNA synthesis, are perhaps the most important Although these pathways have been studied on a macroscopic level, advances in crystallography and biotechnology now make it possible to probe the structure and function of the proteins involved in the control of these pathways at the microscopic level. The understanding of cellular regulation and the proteins involved in the control process will have a great impact on our grasp of cellular differentiation and, as a consequence of this, cures for cancer and birth defects may be found. For the next project period, emphasis will be directed towards two aspects of control of the pyrimidine pathway; first, the enzyme aspartate transcarbamoylase which regulates this pathway by a combination of genetic, metabolic and allosteric control mechanisms; and second, the pyrS gene, the gene-product of which exerts control directly by activating the expression of two of the enzymes of the pathway. The X-ray structures of aspartate transcarbamoylase in the two allosteric forms provides for the first time the necessary structural information to probe this complex system by single amino acid replacements. By the analysis of mutant forms of this enzyme, it will be possible to determine on a functional basis how this enzyme can exert metabolic and allosteric control over pyrimidine biosynthesis. The understanding of this system on the molecular level is particularly important because this system has become a model for the understanding of a diverse number of biological phenomena. The specific aims for this project period are: (1) use the X-ray structures of aspartate transcarbamoylase and functional studies employing site- specific mutants and hybrids to develop and test an integrated model for homotropic cooperativity (allosteric control) and the heterotropic interactions (metabolic control) in this enzyme, (2) determine the molecular level details of the catalytic mechanism of aspartate transcarbamoylase, (3) determine if second sphere interactions are important for catalysis and cooperativity in proteins in general and in aspartate transcarbamoylase in particular, and (4) purify the pyrS gene-product and determine its interaction with the promoters of the pyrimidine pathway.

该项目的长期目标是获得分子水平 了解控制代谢的细胞控制的机制 途径。 特别是，研究涉及的实际蛋白质分子 在调节过程中，以检查分子基础 酶可以调节其自身活性，并确定细胞如何 调节该特定酶的生物合成。 在所有代谢中 素，嘌呤和嘧啶的生物合成，是 DNA合成的同等数量需要，也许是最重要的 尽管这些途径已在宏观级别上进行了研究，但进步 在晶体学和生物技术中，现在可以探测 与这些控制有关的蛋白质的结构和功能 微观水平的途径。 对细胞的理解 调节和控制过程中涉及的蛋白质将具有 对我们对细胞分化的掌握的巨大影响，因此 其中，可以找到癌症和先天缺陷的治疗方法。 在下一个项目时期，重点将针对两个方面 控制嘧啶途径的控制；首先，酶天冬氨酸 跨菌型层，通过遗传的结合来调节这一途径 代谢和变构控制机制；其次，皮尔斯基因， 基因产物通过激活表达直接施加控制 途径的两个酶。 天冬氨酸的X射线结构 两种变构形式的跨加体层首次提供 单一的必要结构信息，以探究该复杂系统 氨基酸替代品。 通过分析该酶的突变形式， 可以在功能基础上确定该酶如何可以 对嘧啶生物合成的代谢和变构控制。 这 在分子水平上了解该系统尤其是 重要，因为该系统已成为理解的模型 多种生物学现象。 该项目期间的具体目的是：（1）使用X射线结构 天冬氨酸经钙化岩和功能研究采用部位 特定的突变体和杂种，以开发和测试一个集成模型 同型合作（变构控制）和异位 该酶中的相互作用（代谢控制），（2）确定 天冬氨酸催化机制的分子水平细节 跨足菌，（3）确定第二球相互作用是否为 一般和蛋白质的催化和协同性很重要 特别是天冬氨酸经氨基甲酰胺层，（4）净化pyrs 基因产品并确定其与启动子的相互作用 嘧啶途径。