THE MOLECULAR BASIS OF CELLULAR CONTROL MECHANISMS

描述（由申请人提供）： 该项目的长期目标是了解生物系统中信号传输的过程。在这个项目期间，我们将集中在两个变构酶，天冬氨酸转氨甲酰酶（ATCase）和果糖1，6-二磷酸酶（FBPase），分别控制嘧啶和葡萄糖生成途径的速率。变构酶的调节涉及信号分子与特定调节位点的结合，这种结合诱导改变其活性的构象变化。ATCase调节参与核酸前体生物合成的嘧啶途径。由于ATCase的抑制阻止细胞增殖，这种酶已成为抗癌药物开发的目标。FBPase参与葡萄糖生成的控制，当这种调节失败时，血糖水平就会改变，这使得这种酶成为开发抗糖尿病药物的目标。对这些控制酶的分子水平的理解将为合理开发药物提供基础，这些药物可在需要时专门用于调节这些途径。该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。 这项建议的具体目标是：（i）使用时间分辨X射线散射来表征ATCase的变构转变和在变构转变期间存在的瞬时稳定中间体，（ii）使用芘标记形式的酶、由一条野生型链和一条不能结合效应物的链组成的杂合调节亚基来研究ATCase中的信号传递机制，和调节链，其中变构和锌结构域通过二硫键形成而构象锁定，（iii）使用杂交体、荧光标记和质谱法区分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射线晶体学来确定调节信号的传递和蛋白质动态运动的作用，例如结构域闭合和调节信号的这种传递的环运动。

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.