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.
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