The long-term objective of this proposal is to define the structure- function relation in the E. coli enzyme, glutamine-dependent carbamyl phosphate synthetase (CPS). This enzyme with a unique mechanism of catalysis synthesizes a high energy precursor (carbamyl phosphate), required for pyrimidine biosynthesis in all living organisms. The protein has a complex mosaic structure with active sites related to the biotin-dependent carboxylases, and to a large family of enzymes that provide amide nitrogen for diverse biosynthetic reactions. At present, very little is known about these multifunctional enzymes and the manner in which their different active sites spatially and temporally interact during conversion of substrate to products.
The specific aims are: (1) to continue the structure determination of E coli CPS by X-ray and multiwavelength anomalous diffraction analysis in a collaborative arrangement with Dr. Wayne Hendrickson's laboratory to solve the crystal structure; (2) to determine the structure of different catalytic and conformational forms of the protein; (3) to identify the functions of essential catalytic residues at each of the two ATP binding sites from hypotheses generated by the structure. Catalytic site mutants impaired in active site coupling are anticipated to provide additional mechanistic details of carbamate synthesis and phosphorylation; (4) to define residues essential to the function of the glutamine hydrolytic site, through site modification and kinetic analysis of thioester and product formation. New insights derived from the three-dimensional structure of CPS bear directly on the field of molecular medicine. Lesions in the human NH3-dependent enzyme either are lethal or lead to hyperammonemia, hepatic coma, and CNS damage. New directed enzyme therapies are continually being developed. Human pyrimidine-specific CPS is highly elevated in rapidly proliferating tumor tissue, and, therefore is a prime candidate for design of potent and selective inhibitors of the rapid tissue growth associated with cancer.
Showing the most recent 10 out of 18 publications
