title: A convenient gHMQC-based NMR assay for investigating ammonia channeling in glutamine-dependent amidotransferases: studies of Escherichia coli asparagine synthetase B.
abstract: X-ray crystal structures of glutamine-dependent amidotransferases in their "active" conformation have revealed the existence of multiple active sites linked by solvent inaccessible intramolecular channels, giving rise to the widely accepted view that ammonia released in a glutaminase site is channeled efficiently into a separate synthetase site where it undergoes further reaction. We now report a very convenient isotope-edited 1H NMR-based assay that can be used to probe the transfer of ammonia between the active sites of amidotransferases and demonstrate its use in studies of Escherichia coli asparagine synthetase B (AS-B). Our NMR results suggest that (i) high glutamine concentrations do not suppress ammonia-dependent asparagine formation in this bacterial asparagine synthetase and (ii) ammonia in bulk solution can react with the thioester intermediate formed during the glutaminase half-reaction by accessing the N-terminal active site of AS-B during catalytic turnover. These observations are consistent with a model in which exogenous ammonia can access the intramolecular tunnel in AS-B during glutamine-dependent asparagine synthesis, in contrast to expectations based on studies of class I amidotransferases.
authors.affiliation: Department of Chemistry, University of Florida, Gainesville, Florida 32611-7200, USA.
authors.forename: Kai K
authors.initials: KK
authors.lastname: Li
authors.forename: William T
authors.initials: WT
authors.lastname: Beeson
authors.forename: Ion
authors.initials: I
authors.lastname: Ghiviriga
authors.forename: Nigel G J
authors.initials: NG
authors.lastname: Richards
mesh_terms: D000641:Ammonia / Q000378:metabolism*; D001217:Aspartate-Ammonia Ligase / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011789:Quantum Theory
chemical_list: D009587:Nitrogen Isotopes; D000641:Ammonia; D001217:Aspartate-Ammonia Ligase