STRUCTURE-ENERGY CORRELATE IN ASPARTATE TRANSCARBAMYLASE

这项研究的长期目标是阐明变构 大肠杆菌天冬氨酸氨基转移酶的作用机制。在过去的工作中 阐述了配体结合和亚基相互作用的热力学。 我们将通过测定自由能来扩展我们的热力学研究。 氢交换测量的链间和亚基间相互作用 和分析凝胶层析，并通过评估热容的变化 结合天然酶和分离亚基的伴随配体 间歇量热法。然而，未来工作的主旨将是 把热力学和结构联系起来，也就是说，来确定 用于调节的能量分布在整个结构中。三 我们将采取各种方法。(A)Gurd-Matthew静态可达性 离散电荷模型或其改进将与 用于识别和表征可电离基团的晶体坐标 受到配体结合和亚基结合的干扰。(B)中等分辨率 氢交换方法将被开发并用于表征 冠状病毒引起的ATCase及其亚基的分子动力学变化 效应器结合和亚单位关联，并识别 结构受到这些相互作用的干扰。(C)单点突变将是 用来识别参与病毒传播的分子区域 变构信号。选定的突变体还将被表征为 关于它们的静电学(通过电位法和计算机 计算)、热稳定性(通过差示扫描量热法)，以及 分子动力学(通过氢交换)。通过以下方式获得的信息 这三种方法将被用来开发一个量化的模型 变构机制。

The long range goal of this research is the elucidation of the allosteric mechanism of E. coli aspartate transcarbamylase. Work in the past has empahsized the thermodynamics of ligand binding and subuint interactions. We will extend our thermodynamic studies by determining free energies of interchain and intersubunit interactions by hydrogen exchange measurements and analytical gel chromatography, and by evaluating heat capacity changes accompanying ligand binding to the native enzyme and isolated subunits by batch calorimetry. The main thrust of future work, however, will be to correlate thermodynamics and structure, that is, to determine how the energy used for regulation is distributed throughout the structure. Three approaches will be taken. (a) The Gurd-Matthew static accessibility discrete charge model, or refinements of it, will be used together with the crystallographic coordinates to identify and characterize ionizable groups perturbed by ligand binding and subunit association. (b) Medium resolution hydrogen exchange methods will be developed and used to characterize changes in the molecular dynamics of ATCase and its subunits produced by effector binding and subunit association, and to identify regions of the structure perturbed by these interactions. (c) Single site mutants will be used to identify regions of the molecule involved in the transmission of allosteric signals. Selected mutants will also be characterized with respect to their electrostatics (by potentiometry and computer calculations), thermostability (by diffeerential scanning calorimetry), and molecular dynamics (by hydrogen exchange). The information obtained with these three approaches will be used to develop a quantitative model of the allosteric mechanism.