DIRECT OBSERVATION OF THE QUATERNARY CONFORMATIONAL CHANGES INDUCED BY SUBSTRATE

直接观察底物引起的四元构象变化

• 批准号: 7722147
• 负责人: EVAN R KANTROWITZ
• 金额: $ 0.02万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722147
• 关键词: Anabolism; Antimalarials; Aspartate; Carbamoyl Transferases; Computer Retrieval of Information on Scientific Projects Database; Crystallography; Enzymes; Funding; Grant; Institution; Metabolic; Metabolism; Molecular; Molecular Conformation; Monitor; Nucleotide Biosynthesis; Pharmaceutical Preparations; Pyrimidine; Pyrimidine Nucleotides; Pyrimidines; Rate; Reaction; Regulation; Research; Research Personnel; Resources; Source; Time; United States National Institutes of Health; beamline; nucleic acid biosynthesis; research study; time use

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project involves the study of a critical enzyme in metabolism, aspartate transcarbamoylase (ATCase). ATCase catalyzes the first step in pyrimidine nucleotide biosynthesis. The product of the reaction, carbamoyl aspartate is then converted into the pyrimidine nucleotides necessary for nucleic acid biosynthesis. ATCase has been identified as a target for anti-proliferation and anti-malarial drugs. Particularly important is that ATCase not only catalyzes the above reaction, but also controls the rate of pyrimidine biosynthesis. Regulation is achieved by a conformational switch from a low-activity T-state to a high-activity R-state. These two states have different quaternary conformations that can be easily distinguished by SAXS. By using a stopped flow mixer attached to the SAXS apparatus at SSRL we are ability to monitor the actual transition of the enzyme from the T to the R, and from the R to T, states induced by the natural substrates as well as potential drug candidates. For this project period we have two specific aims: (i) investigate the heterotropic interactions and homotropic cooperativity of ATCase using time-resolved SAXS, and (ii) monitor the cooperativity transition of ATCase from the T to the R state by time-resolved crystallography. The first specific aim is directed at determining the molecular level details of how ATCase is able to regulate pyrimidine nucleotide biosynthesis. The second specific aim will utilize the new capabilities of beamline 4-2 to obtain a time-lapsed record of the conformational changes that are required to convert the enzyme from the T to the R state by x-ray crystallography. This experiment will then be combined with other ongoing studies to determine by crystallography each of the steps in the catalytic and regulatory mechanisms of this important metabolic enzyme.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 该项目涉及代谢中关键酶天冬氨酸转氨甲酰酶（ATCase）的研究。 ATCase 催化嘧啶核苷酸生物合成的第一步。然后反应产物氨基甲酰天冬氨酸转化为核酸生物合成所需的嘧啶核苷酸。 ATCase 已被确定为抗增殖和抗疟疾药物的靶标。特别重要的是，ATCase不仅催化上述反应，还控制嘧啶生物合成的速率。调节是通过从低活性 T 状态到高活性 R 状态的构象转换来实现的。这两种状态具有不同的四级构象，可以通过 SAXS 轻松区分。通过使用连接到 SSRL 的 SAXS 装置的停流混合器，我们能够监测酶从 T 到 R 以及从 R 到 T 的实际转变，以及由天然底物和潜在候选药物诱导的状态。在这个项目期间，我们有两个具体目标：(i) 使用时间分辨 SAXS 研究 ATCase 的异向相互作用和同向协同性，以及 (ii) 通过时间分辨晶体学监测 ATCase 从 T 态到 R 态的协同性转变。第一个具体目标是确定 ATCase 如何调节嘧啶核苷酸生物合成的分子水平细节。第二个具体目标是利用光束线 4-2 的新功能，获得通过 X 射线晶体学将酶从 T 态转化为 R 态所需的构象变化的延时记录。然后，该实验将与其他正在进行的研究相结合，通过晶体学确定这种重要代谢酶的催化和调节机制的每个步骤。