Research Project
7574788
[unreadable] DESCRIPTION (provided by applicant): The long-term objective for the research described in this application is aimed at the elucidation of the physical chemical relationships between structure and function in biological systems. The primary focus of this proposal will be directed towards a fundamental understanding of chemical reaction mechanisms and allosteric interactions among the multiple active sites within the amidotransferase family of proteins. This group of enzymes catalyzes the formation of amide functional groups in substrates using ammonia/glutamine as the nitrogen source and ATP as the activating agent. All amidotransferases that use glutamine as a nitrogen source require separate active sites for the generation and utilization of ammonia. These distinct active sites are between 10-45 A apart and are connected by an intramolecular tunnel for the passage of ammonia from one site to the next. The reactions catalyzed by members of the amidotransferase superfamily are critical for the biosynthesis of carbohydrates, nucleic acids, amino acids and essential coenzymes and thus these enzymes are attractive targets for pharmaceutical intervention and regulation. The three enzymes to be examined in this proposal are carbamoyl phosphate synthetase (CPS), cobyric acid synthetase (CbiP), and asparagine synthetase A (AsnA). CPS is an essential enzyme found in all organisms where it catalyzes the first committed step in the biosynthesis of pyrimidine nucleotides and in the detoxification of ammonia via the urea cycle. The synthesis of carbamoyl phosphate by a single protein is one of the more complicated reactions in biological chemistry inasmuch as five substrates are converted into five separate reaction products. The overall reaction mechanism and ligand induced conformational changes will be determined via a combination of kinetic measurements, fluorescence spectroscopy, molecular dynamics simulations, and characterization of site directed mutants. Cobyric acid synthetase is responsible for the amidation of four separate carboxylate groups attached to the corrin ring system during the biosynthesis of coenzyme B12. Cobyric acid synthetase functions to form amides from carboxylates b, d, e, and g in coenzyme B12 via a mechanism that is apparently ordered and dissociative. The structural basis for the unusual reaction specificity and the allosteric coupling between the multiple active sites will be determined by the isolation of reaction intermediates, pre-steady state kinetic studies and macromolecular structure elucidation. Asparagine synthetase A is known to catalyze the formation of asparagine from ATP, ammonia, and aspartic acid. However, this enzyme has recently been implicated in the biosynthesis of an extracellular death factor peptide, NNWNN, in Escherichia coli. The role of AsnA in the formation of this very unusual peptide will be determined. PUBLIC HEALTH RELEVANCE: The focus of this application will be directed at a determination of the role that three enzymes play in the biosynthesis of pyrimidine nucleotides, vitamin B12, and a short peptide that initiates the killing of bacteria. These studies may lead to the development of new antibiotics in the defense of bacterial infections. [unreadable] [unreadable] [unreadable]
