Control mechanisms are of crucial importance for the maintenance of normal metabolism and a knowledge of the molecular details of the mechanisms that regulate metabolism is essential for elucidation of pathological processes. Thus, knowledge of the molecular mechanisms for regulation of the urea cycle, the major pathway of ammonia removal, will prove an understanding of disorders (e.g., Reye's syndrome, hepatitis, cirrhosis and metabolic defects) in which liver function is temporarily or permanently altered. Carbamoyl-phosphate synthetase (CPS), which catalyzes the entry step of the urea cycle, is the primary site of control under many physiological conditions. CPSs vary in subunit structure, nitrogen donor (free ammonia and/or ammonia derived from glutamine), and regulators. However, all CPSs have similar sequences folded into a similar multi-domain structure, and all appear to use a common mechanism to catalyze the form of CP, P/i and 2 ADP from HCO/3, NH/3 and 2 ATP. For glutamine-dependent CPSs, subdomain A-2 is a glutaminases; ammonia- specific CPSs retain this region but with replacement of essential residues. Subdomain A-1 mediates interaction between the glutaminase and synthetase moieties. Domain D is the regulatory domain where binding of many allosteric effectors occurs. The two molecules of ATP are utilized at two internally duplicated domains, B and C, with domain D possibly folding to facilitate ATP utilization. The recently reported crystal structure for CPS has shown that domains B and C are structurally equivalent to each other and to the other four members of the N-ligase structural family. We have proposed a novel mechanism for CPS, which couples ATP binding and hydrolysis at domain C to domain B cycling between two alternative conformations. Our first three specific aims are intended to test specific, distinguishing features of the proposed reaction scheme, but will yield significant insights in the structure/function relationship for CPS regardless of the pathway utilized. Proposed specific aims 4 and 5 are intended to elucidate the structural and functional routes of communication among the various domains of CPS that allow its catalysis and regulation.
