The long term objectives of this project are to acquire a better understanding of one extremely important cellular control mechanism - the regulation of the rates of metabolic pathways by allosteric enzymes. Allosteric enzymes can be considered the most important type of enzymes, because they carry two functions - they both catalyze a reaction in a metabolic pathway, and also control the rate of the entire pathway. Regulation by allosteric enzymes involves the binding of signaling molecules to specific regulatory sites on the enzyme; this binding induces an alteration in catalytic activity. We have chosen for investigation the allosteric enzyme aspartate transcarbamoylase which catalyzes the first step in the pyrimidine pathway. This pathway is critical because the end products, the pyrimidine nucleotides, are necessary for DNA synthesis, and therefore it has become a common target for antiproliferation drugs. Aspartate transcarbamoylase regulates the pyrimidine biosynthesis pathway, but even more importantly, it has become a model system for the understanding of a diverse number of biological phenomena. For a comprehensive understanding of aspartate transcarbamoylase we must elucidate (i) the manner by which the enzyme accelerates the reaction rate, (ii) the manner by which the enzyme shifts between the """"""""off"""""""" and """"""""on"""""""" states, and (iii) the manner by which the activity of the enzyme is altered by the binding of the regulatory molecules 60 Angstrom units from the active site. We have previously proposed molecular mechanisms for each of these actions; here we propose to test them on both the functional and structural levels. The goals for this project period are to: (i) determine whether intermediate states exist during the allosteric transition of the wild-type enzyme; (ii) map the structural transition using intermediates along the path between the T and R states; (iii) determine the energetic contribution of T and R-state stabilizing interactions for both homotropic cooperativity and the heterotropic effects; (iv) investigate the enzyme-substrate complex by determining structural details of the active site and the quaternary structure in the presence of the natural substrates and substrate analogs; and (v) determine the mode of action of the heterotropic effectors using X-ray crystallography and small-angle X-ray scattering.
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