DIRECT OBSERVATION OF THE QUATERNARY CONFORMATIONAL CHANGES INDUCED BY SUBSTRATE

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 本计画研究代谢中的关键酵素--天冬氨酸转氨甲酰酶（ATCase）。ATCase催化嘧啶核苷酸生物合成的第一步。反应产物氨基甲酰天冬氨酸随后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点。特别重要的是，ATCase不仅催化上述反应，而且还控制嘧啶生物合成的速率。通过从低活性T状态到高活性R状态的构象转换来实现调节。这两种状态具有不同的四元构象，可以很容易地区分SAXS。通过在SSRL使用连接到SAXS装置的停流混合器，我们能够监测由天然底物以及潜在的候选药物诱导的酶从T到R以及从R到T状态的实际转变。在这个项目期间，我们有两个具体的目标：（i）调查异向性相互作用和同向性协同ATCase使用时间分辨SAXS，和（ii）监测协同ATCase从T到R状态的时间分辨晶体学。第一个具体目标是确定ATCase如何能够调节嘧啶核苷酸生物合成的分子水平细节。第二个具体目标将利用光束线4-2的新功能，通过X射线晶体学获得将酶从T状态转化为R状态所需的构象变化的时间推移记录。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中的每个步骤。

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.