Regulation of enzyme synthesis and control of activity of preexisting enzymes are two basic mechanisms for adjusting the rates of metabolic reactions in the living cell. The long-term objective of this research is to explore the molecular basis of enzyme regulation in intact cells and in isolated, purified systems. The biodegradative the ronine dehydratase, an enzyme involved in anaerobic energy metabolism and subject to multilevel controls, is an ideal prototype model system to analyze the structure and control of gene expression, and the regulatory interactions between enzymes and cellular metabolites that alter the conformation and the catalytic potential of the enzyme. In the proposed research two parallel experimental strategies-biochemical studies with purified protein and a molecular genetic analysis of the cloned DNA-will be adopted to determine the complete nucleotide sequence of the dehydratase gene, examine its control region and deduce the primary structure of the enzyme; identify the active and regulatory sites by covalent ligand binding and affinity labeling experiments to map their respective locations on the protein structure; and isolate regulatory mutants by operon-fusion technique and transposon mutagenesis to examine the factors and mechanisms involved in anaerobic enzyme induction by amino acids, cAMP, and electron acceptors. These studies will help elucidate the molecular architecture of a regulatory enzyme and its regulation by cellular metabolites. It is hoped that this new knowledge on biological control mechanisms may provide insight into the basic metabolic processes in the living cell and how loss of normal regulatory controls may trigger metabolite imbalance and biochemical disorders.
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