ALLOSTERISM IN E COLI CARBAMOYL PHOSPHATE SYNTHETASE

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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