Revisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli.
Escherichia coli asparagine synthetase B (AS-B) catalyzes the formation of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the in vivo nitrogen source. In an effort to reconcile several different kinetic models that have been proposed for glutamine-dependent asparagine synthetases, we have used numerical methods to investigate the kinetic mechanism of AS-B. Our simulations demonstrate that literature proposals cannot reproduce the glutamine dependence of the glutamate/asparagine stoichiometry observed for AS-B, and we have therefore developed a new kinetic model that describes the behavior of AS-B more completely. The key difference between this new model and the literature proposals is the inclusion of an E.ATP.Asp.Gln quaternary complex that can either proceed to form asparagine or release ammonia through nonproductive glutamine hydrolysis. The implication of this model is that the two active sites in AS-B become coordinated only after formation of a beta-aspartyl-AMP intermediate in the synthetase site of the enzyme. The coupling of glutaminase and synthetase activities in AS is therefore different from that observed in all other well-characterized glutamine-dependent amidotransferases.
Tesson
Alan R
AR
Department of Chemistry, University of Florida, Gainesville 32611, USA.
Soper
Thomas S
TS
Ciustea
Mihai
M
Richards
N G J
NG
D000641:Ammonia / Q000378:metabolism; D001216:Asparagine / Q000096:biosynthesis; D001217:Aspartate-Ammonia Ligase / Q000737:chemistry / Q000378:metabolism; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D011487:Protein Conformation
D005973:Glutamine; D001216:Asparagine; D000641:Ammonia; D001217:Aspartate-Ammonia Ligase; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; C019742:asparagine synthetase (glutamine-hydrolyzing)