This research will concentrate on the understanding of the molecular basis of enzyme action. In particular, to study the actual protein molecules involved in the regulatory process and to examine the molecular basis by which an enzyme can regulate its own activity and how the cell can regulate the biosynthesis of that particular enzyme. The understanding of cellular regulation would have a great impact on our grasp of cellular differentiation and as a consequence of this cures for cancer and birth defects may be found. Emphasis will be directed towards two aspects of control of the pyrimidine biosynthesis pathway, the products of which are necessary for DNA replication. First, the enzyme aspartate transcarbamylase which regulates this pathway by a combination of genetic, metabolic and allosteric control mechanisms; and second, the pyrS gene, a new gene which we recently discovered, which is directly involved in control of this pathway. In addition, we will use E. coli alkaline phosphatase as a model to study the in vitro modification of enzyme activity. After the analysis of our current set of over 200 mutant versions of aspartate transcarbamylase with single amino acid substitutions is completed, we will generate specific mutants by in vitro mutagenesis to acquire sufficient information to propose a mechanism for the homotropic and heterotropic interactions of the enzyme. After the genetic characterization of the pyrS gene is completed, we will further characterize the pyrS gene-product. We will also investigate the interaction of the pyrS gene-product with its metabolic effector and its DNA binding site, in order to determine the mechanism by which it exerts control over the pyrimidine pathway. Finally, will be used in vitro mutagenesis to delineate the catalytic mechanism and cooperativity of alkaline phosphatase and to use these techniques to alter the specificity and catalytic efficiency of the enzyme. The use of recombinant DNA technology will substantially increase our ability to answer these fundamental questions and, therefore, the specific aim of this Research Career Development Award is to provide me an opportunity to integrate this new technology into my research.
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