MECHANISMS OF SYNCHRONIZATION: VESTIGIAL PATHS TO UREA CYCLE DEFICIENCIES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这项研究的长期目标是解决蛋白质间变构通讯的机制，蛋白质是多聚体酶和酶复合体调节、配体诱导的寡聚和同步化的常见生化基序。这类系统的一个范例是来自大肠杆菌的氨基甲酰磷酸合成酶(CPS)。该酶通过五种底物、至少三个不稳定的中间体和三个相隔近100°的不同活性部位合成氨基甲酰磷酸，随后被同化为精氨酸和各种嘧啶。随着反应物之间实现了完美的化学计量比，反应中心的同步被认为最初是通过小亚基和大亚基之间的变构冲动进行的。因此，这项建议的具体目标是：(1)利用固有的荧光团探测蛋白质基质的特定区域，以定位和表征动态的构象变化，建立亚基间的同步机制；以及(2)评估底物、过渡态和活性中间体的类似物结合的潜力，以诱导同步的动力学信号和/或构象变化，从而突出活性位点协调的潜在“触发器”。主要技术将包括：细菌系统内的定点突变；通过多个耦合酶系统的酶动力学；以及稳态和频域荧光光谱。更广泛地说，在将蛋白质催化域的编排与其动态构象变化相关联时，也将深入了解整个酶系统如何在共享不稳定的中间产物时整合在一起。

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.