DIRECT OBSERVATION OF THE QUATERNARY CONFORMATIONAL CHANGES INDUCED BY SUBSTRATE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该项目涉及新陈代谢中的一种关键酶--天冬氨酸氨基转移酶(ATCase)的研究。ATCase催化嘧啶核苷酸生物合成的第一步。然后，该反应的产物氨基甲酰天冬氨酸被转化为核酸生物合成所必需的嘧啶核苷酸。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，而且还控制着嘧啶的生物合成速度。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元构象，可以通过SAXS很容易地区分开来。通过在SSRL的SAXS设备上使用停流混合器，我们能够监测酶从T到R，从R到T的实际转变，天然底物诱导的状态以及潜在的候选药物。在这个项目期间，我们有两个具体的目标：(I)用时间分辨SAXS研究ATCase的各向异性相互作用和各向同性协同作用；(Ii)用时间分辨结晶学监测ATCase从T态到R态的协同性转变。第一个具体目标是确定ATCase如何能够调控嘧啶核苷酸生物合成的分子水平细节。第二个具体目标将利用光束线4-2的新能力来获得通过X射线结晶学将酶从T状态转变为R状态所需的构象变化的时间推移记录。然后，这项实验将与其他正在进行的研究相结合，通过结晶学来确定这种重要代谢酶的催化和调节机制中的每一步。

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.