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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 该项目涉及对新陈代谢，天冬氨酸型经钙化岩（ATCASE）中关键酶的研究。 Atcase催化嘧啶核苷酸生物合成的第一步。反应的产物，然后将甲状腺氨甲酸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。尤其重要的是，Atcase不仅催化上述反应，还可以控制嘧啶生物合成的速率。调节是通过从低活动性t状态到高活动性R状态的构象开关来实现的。这两个状态具有不同的第四纪构象，很容易被萨克斯群体区分。通过使用SSRL处的SAXS设备上的停止流动混合器，我们可以监测酶从T到R的实际过渡到R，以及从自然基质引起的状态以及潜在的药物候选物引起的状态。在这个项目期间，我们有两个具体的目的：（i）使用时间分辨的萨克斯公司（SAXS）研究ATCASE的异型相互作用和同型合作，以及（ii）通过时间分辨的晶体学来监测ATCASE从T到R状态的合作过渡。第一个具体目的是确定ATCASE如何调节嘧啶核苷酸生物合成的分子水平细节。第二个特定目的将利用光束线4-2的新功能获得通过X射线晶体学从T将酶从T转换为R状态所需的构象变化的记录。然后，该实验将与其他正在进行的研究结合使用，以通过晶体学确定这种重要的代谢酶的催化和调节机制中的每个步骤。