MAPPING NUCLEOTIDE SITES OF E. COLI GMP SYNTHETASE

Lebanon Valley College, Annville PA

Investigators

Abstract

DESCRIPTION (from applicant's abstract): This project seeks to elucidate amino acid residues involved in nucleotide binding and catalysis in the ATP pyrophosphatase domain of guanosine-5'-monophosphate synthetase (GMPS) from E. coli. GMPS catalyzes the terminal step in the de novo pathway for the biosynthesis of guanosine nucleotides and, as such, is a target enzyme for chemotherapeutic and immunosuppressive therapies. The GMPS monomer is organized into two independently folded domains, each of which is related by sequence and function to larger enzyme families. The ATP pyrophosphatase domain provides binding sites for the nucleotides ATP and XMP and catalyzes the formation of an adenyl-XMP intermediate that reacts with ammonia produced on the glutamine amidotransferase domain, forming the product GMP. The experimental strategy relies on two complementary approaches: site-directed mutagenesis and affinity labeling. An analysis of crystal structure and sequence has targeted 18 amino acids for mutagenic substitution. Characterization of constructed mutants by enzyme kinetics and ligand binding measurements will provide data necessary to evaluate the roles of the targeted amino acids in catalysis and ligand binding. Affinity labeling by reactive analogs of guanosine and adenosine nucleotides, a technique that can independently target both purine and 5'-phosphate regions of each nucleotide binding site, will be used to covalently label amino acid residues at the two sites. MALDI-TOF mass spectrometry will confirm covalent labeling of the modified proteins, and a process involving proteolysis, peptide separation by HPLC, and peptide analysis by MALDI-TOF and Edman sequencing will be used to identify amino acids that react with the affinity labels. Affinity labeling is expected both to confirm some of the residues elucidated by mutagenesis and to identify other residues not originally selected for mutagenesis. Amino acids elucidated in this project will contribute to a detailed active site mapping of the nucleotide binding domain of GMPS, an enzyme that is both a target for the design of pharmaceutical agents and a representative member of wider enzyme families.