The broad long term objectives of this proposal are directed at understanding the relationship between structure and function in biological systems. This application is targeted at carbamoyl phosphate synthetase (CPS); an enzyme that is responsible for the production of the key intermediate during the biosynthesis of arginine (via the urea cycle) and the pyrimidine nucleotides in all types of cells.
The specific aims of this proposal include the following: (1) The chemical reaction catalyzed by this enzyme is quite complex since five substrates are converted to five separate products. The free energy profiles, and spectroscopic identification of reaction intermediates (carboxyphosphate, carbamate, carbon dioxide, and gamma-glutamyl thioester) will be determined by a coordinated program of steady state, rapid quench, and NMR experimental protocols. (2) The bacterial enzyme is composed of two subunits of unequal size. The elucidation of the functional domain boundaries and the role of individual amino acids in binding, catalysis, and regulatory control of this enzyme will be determined by the design, construction, and functional characterization of site-directed and randam mutants. (3) The large synthetase subunit contains a large internal tandem repeat of approximately 400 residues. The functional and structural differences between these two homologous domains will be determined by the creation and characterization of chimeric tandem repeats of either the N-terminal or C-terminal portion of the wild type enzyme. (4) The coordinated control of the reaction intermediates within this enzyme serves as a well-behaved model system for the channeling of reaction products between successive enzymes within a metabolic pathway. The relationship between the binding sites for the production of carbamoyl phosphate will be determined by the elucidation of the three dimensional structure of the enzyme by x-ray diffraction methods on single protein crystals.
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