THE MOLECULAR BASIS OF CELLULAR CONTROL MECHANISMS

细胞控制机制的分子基础

• 批准号: 3273743
• 负责人: EVAN R KANTROWITZ
• 金额: $ 20.16万
• 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/3273743
• 关键词: DNA replication; X ray crystallography; active sites; allosteric site; aspartate carbamoyltransferase; chemical kinetics; chemical stability; chemical substitution; circular dichroism; conformation; 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 了解细胞代谢控制的机制 途径。 特别是研究所涉及的实际蛋白质分子 在监管过程中，检查分子基础 酶可以调节自身的活性，并决定细胞如何 调节该特定酶的生物合成。 在所有的新陈代谢中 嘌呤和嘧啶的生物合成途径 DNA 合成所需的等量，也许是最重要的 尽管这些途径已经在宏观层面上进行了研究，但进展 晶体学和生物技术现在使得探索 参与控制这些的蛋白质的结构和功能 微观层面的途径。 对细胞的理解 调节和参与控制过程的蛋白质将具有 对我们对细胞分化的理解产生了巨大影响，因此 其中，可能会找到治疗癌症和出生缺陷的方法。 下一个项目期间，重点将集中在两个方面 嘧啶途径的控制；首先，天门冬氨酸酶 转氨甲酰酶通过遗传、 代谢和变构控制机制；第二，pyrS 基因， 其基因产物通过激活表达直接发挥控制作用 该途径的两种酶。 天门冬氨酸的X射线结构 两种变构形式的转氨甲酰酶首次提供 必要的结构信息来探测这个复杂的系统 氨基酸替代品。 通过对该酶的突变形式的分析， 将有可能在功能基础上确定这种酶如何发挥作用 对嘧啶生物合成发挥代谢和变构控制。 这 对这个系统在分子水平上的理解尤其重要 很重要，因为这个系统已经成为理解的模型 多种生物现象。 本项目期的具体目标是：（1）利用X射线结构 天冬氨酸转氨甲酰酶的研究和使用位点的功能研究 特定突变体和杂交体来开发和测试集成模型 同向协同性（变构控制）和异向性 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 转氨甲酰酶，(3) 确定第二球体相互作用是否 对于一般蛋白质和在蛋白质中的催化和协同作用很重要 特别是天冬氨酸转氨甲酰酶，并且（4）纯化pyrS 基因产物并确定其与启动子的相互作用 嘧啶途径。