ALLOSTERISM IN E COLI CARBAMOYL PHOSPHATE SYNTHETASE

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。一个变构网络的相互作用协调活性位点内的氨甲酰磷酸合成酶（CPS）从大肠杆菌。大肠杆菌实现反应物和产物之间精确的化学计量，以及通过激活剂鸟氨酸和抑制剂UMP的结合来调节活性位点内的活性。为了分离和量化建立这种通信网络的变构相互作用，我们设计了一系列CPS变体，每个变体包含六个天然的Dahans之一，作为构象变化的荧光探针。结果表明，ATP结合在大亚基诱导可测量的变化，在小亚基的W170和W175的荧光响应，也许提供洞察构象变化同步的前两个反应中心的CPS。此外，两个ATP结合结构域的突变研究表明，它是ATP结合内的羧基磷酸结构域，特异性地诱导W213的荧光强度的戏剧性的40%的增加。我们已经利用这种变化来表征在0.2mg/mL的CPS浓度下ATP的Kd作为变构调节剂的函数，揭示了偶联（即，一种配体的结合对第二种配体的结合亲和力的影响），其模拟在0.001 mg/mL下动力学测定的那些。然而，粗略分析1.6 mg/mL（已知存在较高CPS聚集体的浓度，但稳态动力学实验无法达到）的ATP/鸟氨酸和ATP/UMP偶联显示出真实的差异。未来的分析将集中在：（1）完成每个单色氨酸变体的荧光表征（丙烯酰胺猝灭和偏振研究）;（2）量化CPS聚集状态下羧基磷酸结构域内变构偶联的依赖性;（3）开发对氨甲酰磷酸结构域内ATP结合特异敏感的独特荧光探针。

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. An allosteric web of interactions coordinates active sites within carbamoyl phosphate synthetase (CPS) from E. coli to realize a precise stoichiometry between reactants and products, as well as to modulate activities within the active sites via the binding of the activator ornithine and inhibitor UMP. To isolate and quantify the allosteric interactions establishing this communication network, we have engineered a series of CPS variants, each containing one of the six native tryptophans, to serve as fluorescent probes into conformational change. Results indicate that ATP binding in the large subunit induces measurable changes in the fluorescence response of both W170 and W175 in the small subunit, perhaps providing insight into conformational changes synchronizing the first two reaction centers of CPS. In addition, mutational studies of both ATP binding domains suggest it is ATP binding within the carboxyphosphate domain that specifically induces a dramatic 40% increase in the fluorescence intensity of W213. We have utilized this change to characterize the Kd for ATP as a function of allosteric regulators at a CPS concentration of 0.2mg/mL, revealing couplings (i.e., the impact that the binding of one ligand has on the binding affinity of a second ligand) that mimic those determined kinetically at 0.001mg/mL. However, cursory analysis of ATP/ornithine and ATP/UMP couplings at 1.6mg/mL (a concentration at which higher aggreagates of CPS are known to exist, but which are not experimentally attainable with steady-state kinetics) show real differences. Future analyses will concentrate on: (1) completing the fluorescence characterization (acrylamide quenching and polarization studies) of each single-tryptophan variant; (2) quantifying the dependence of allosteric couplings within the carboxyphosphate domain on the aggregation state of CPS; and (3) developing unique fluorescence probes specifically sensitive to ATP binding within the carbamoyl phosphate do