MECHANISMS OF SYNCHRONIZATION: VESTIGIAL PATHS TO UREA CYCLE DEFICIENCIES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 本研究的长期目标是解决跨蛋白质的变构通信机制，这是调节、配体诱导的寡聚化和多聚体酶以及酶复合物的同步化中的常见生化基序。 这类系统的范例是来自大肠杆菌的氨甲酰磷酸合成酶（CPS）。杆菌 这种酶合成氨甲酰磷酸，随后同化成精氨酸和各种嘧啶，通过一种机制，采用五种底物，至少三种不稳定的中间体，和三个不同的活性位点，相隔近100？。 在反应物之间实现完美的化学计量时，反应中心的同步被认为最初通过小亚基和大亚基之间的变构脉冲进行。 因此，该建议的具体目标是：（1）用内在荧光团探测蛋白质基质的特定区域，以定位和表征建立亚基间同步化机制的动态构象变化;和（2）评估底物、过渡态和反应性中间体的类似物结合以诱导同步动力学信号和/或构象变化的潜力，从而突出了主动站点协调的潜在“触发器”。 主要技术包括：细菌系统内的定点诱变;通过多偶联酶系统的酶动力学;以及稳态和频域荧光光谱。 更一般地说，在将蛋白质的催化结构域的编排与其动态构象变化相关联时，还将深入了解整个酶系统如何在共享不稳定中间体的过程中整合在一起。

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. The long-term objective of this research is to resolve mechanisms of allosteric communications across proteins, a common biochemical motif in the regulation, ligand-induced oligomerization, and synchronization of multimeric enzymes as well as enzyme complexes. A paradigm for such systems is carbamoyl phosphate synthetase (CPS) from E. coli. This enzyme synthesizes carbamoyl phosphate for subsequent assimilation into arginine and the various pyrimidines through a mechanism employing five substrates, at least three unstable intermediates, and three distinct active sites separated by almost 100¿. With a perfect stoichiometry realized between reactants, the synchronization of the reaction centers is thought to proceed initially via an allosteric impulse between the small and large subunits. The specific aims for this proposal are consequently: (1) to probe specific regions of the protein matrix with intrinsic fluorophores to locate and characterize the dynamic, conformational variations establishing the inter-subunit synchronization mechanism; and (2) to assess the potential for the binding of analogs of substrate, transition-state, and reactive intermediate to induce synchronizing kinetic signals and/or conformational changes, thereby highlighting the potential "trigger" for active site coordination. Primary techniques will include: site-directed mutagenesis within a bacterial system; enzyme kinetics via multiple coupling enzyme systems; and steady-state and frequency-domain fluorescence spectroscopy. More generally, in correlating the orchestration of catalytic domains of the protein with its dynamic conformational changes, insight will also be gained into how entire enzyme systems are integrated together in sharing unstable intermediates.