STRUCTURE-ENERGY CORRELATE IN ASPARTATE TRANSCARBAMYLASE

这项研究的长期目标是阐明变构作用， 机制E.大肠杆菌天冬氨酸转氨甲酰酶。 过去的工作 强调了配体结合和亚基相互作用的热力学。 我们将通过确定以下物质的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 和分析凝胶色谱法，并通过评估热容量的变化 伴随配体结合天然酶和分离的亚基， 间歇量热法 然而，今后工作的主要重点将是 关联热力学和结构，也就是说，以确定如何 用于调节的能量分布在整个结构中。 三 将采取办法。 (a)Gurd-Matthew静态可达性 离散电荷模型，或其改进，将与 晶体学坐标来识别和表征可电离基团 受配体结合和亚基缔合的干扰。 (b)中等分辨率 氢交换方法将被开发并用于表征 ATCase及其亚基产生的分子动力学变化 效应子结合和亚基缔合，并鉴定所述亚基的区域。 被这些相互作用扰乱的结构。 (c)单位点突变体将是 用于识别参与传递的分子区域， 变构信号 选择的突变体也将被表征为 关于它们的静电（通过电位测定法和计算机 热稳定性（通过毛细管扫描量热法），和 分子动力学（通过氢交换）。 获得的信息， 这三种方法将被用来开发一个定量模型， 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.