THE MOLECULAR BASIS OF CELLULAR CONTROL MECHANISMS

描述(由申请人提供)： 这个项目的长期目标是了解生物系统中信号传输所涉及的过程。在这个项目期间，我们将专注于两种变构酶，天冬氨酸转氨甲酰酶(ATCase)和果糖1，6-二磷酸酶(FBPase)，它们分别控制嘧啶和糖异生途径的速率。变构酶的调节涉及信号分子与特定调控位点的结合，这种结合诱导构象变化，从而改变它们的活性。ATCase调节与核酸前体生物合成有关的嘧啶途径。由于ATCase的抑制抑制了细胞的增殖，该酶已成为抗癌药物开发的靶点。FBPase参与糖异生的控制，当这一调控失败时，血糖水平会发生变化，使该酶成为抗糖尿病药物开发的靶点。对这些控制酶的分子水平的了解将为合理开发药物提供基础，这些药物可以在需要时特别用于调节这些途径。这个项目直接解决了信号传递的基本问题，特别是酶活性的变构调节。 这个建议的具体目的是：(I)使用时间分辨的X射线散射来表征ATCase的变构转变和在变构转变中存在的瞬时稳定的中间体，(Ii)使用芘标记版本的酶、由一个野生型链和一个不能结合效应器的链组成的杂交调节亚单位以及其中变构和锌结构域通过二硫键构象锁定的调节链来研究ATCase中信号传递的机制，(Iii)使用杂交物、荧光标记和质谱学来区分FBPase中信号传递的局部和全局路径，以表征连接状态的分布，和(Iv)使用X射线结晶学来确定调控信号的传递和蛋白质动态运动的作用，例如结构域关闭和环运动，以实现调控信号的这种传递。

DESCRIPTION (provided by applicant): The long term objectives of this project are to understand the processes involved in signal transmission in biological systems. During this project period we will concentrate on two allosteric enzymes, aspartate transcarbamoylase (ATCase) and fructose 1,6-bisphosphatase (FBPase) that control the rates of the pyrimidine and gluceoneogenesis pathways, respectively. Regulation by allosteric enzymes involves the binding of signaling molecules to specific regulatory sites, and this binding induces conformational changes that alter their activity. ATCase regulates the pyrimidine pathway, which is involved in the biosynthesis of the nucleic acid precursors. Since inhibition of ATCase prevents cell proliferation, this enzyme has become a target for the development of anti-cancer drugs. FBPase is involved in the control of gluceoneogenesis, and when this regulation fails altered blood sugar levels result, making this enzyme a target for the development of anti-diabetic drugs. A molecular level understanding of these control enzymes will provide a basis for the rational development of drugs that can be used to regulate these pathways specifically when required. This project directly addresses fundamental questions of signal transmission in general, and allosteric regulation of enzymatic activity in particular. The specific aims of this proposal are to: (i) use time-resolved X-ray scattering to characterize the allosteric transition of ATCase and the transiently-stable intermediate that exists during the allosteric transition, (ii) investigate the mechanism of signal transmission in ATCase using a pyrene-labeled version of enzyme, hybrid regulatory subunits composed of one wild-type chain and one chain unable to bind effectors, and regulatory chains in which the allosteric and zinc domains are conformationally locked by disulfide bond formation, (iii) differentiate between local and global paths of signal transmission in FBPase using hybrids, fluorescence labels, and mass spectrometry to characterize the distribution of ligated states, and (iv) use x-ray crystallography to determine the transmission of the regulatory signal and the role of protein dynamic motions, such as domain closure, and loop movements for this transfer of the regulatory signal.