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 the 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 glutamine as the nitrogen source and ATP as the activating agent. All amidotransferases examined to date utilize separate active sites for the generation and utilization of ammonia. These distinct active sites are between 10 and 45 A apart and 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 two enzymes to be examined in this proposal are carbamoyl phosphate synthetase (CPS) and cobyric acid synthase (CbiP), an enzyme involved in the biosynthesis of coenzyme B12. 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 reaction mechanism and ligand induced conformational changes will be determined via a combination of kinetic measurements, x-ray crystallography and characterization of site directed mutants of CPS. Cobyric acid synthase is responsible for the amidation of four separate carboxylate groups attached to the corrin ring system of coenzyme B12. Cobyric acid synthase 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 putative active sites will be determined by the direct isolation of reaction intermediates, presteady state kinetic studies and macromolecular structure elucidation.