Restricted passage of reaction intermediates through the ammonia tunnel of carbamoyl phosphate synthetase.
The x-ray crystal structure of the heterodimeric carbamoyl phosphate synthetase from Escherichia coli has identified an intermolecular tunnel that connects the glutamine binding site within the small amidotransferase subunit to the two phosphorylation sites within the large synthetase subunit. The tunneling of the ammonia intermediate through the interior of the protein has been proposed as a mechanism for the delivery of the ammonia from the small subunit to the large subunit. A series of mutants created within the ammonia tunnel were prepared by the placement of a constriction via site-directed mutagenesis. The degree of constriction within the ammonia tunnel of these enzymes was found to correlate to the extent of the uncoupling of the partial reactions, the diminution of carbamoyl phosphate formation, and the percentage of the internally derived ammonia that is channeled through the ammonia tunnel. NMR spectroscopy and a radiolabeled probe were used to detect and identify the enzymatic synthesis of N-amino carbamoyl phosphate and N-hydroxy carbamoyl phosphate from hydroxylamine and hydrazine. The kinetic results indicate that hydroxylamine, derived from the hydrolysis of gamma-glutamyl hydroxamate, is channeled through the ammonia tunnel to the large subunit. Discrimination between the passage of ammonia and hydroxylamine was observed among some of these tunnel-impaired enzymes. The overall results provide biochemical evidence for the tunneling of ammonia within the native carbamoyl phosphate synthetase.
Huang
X
X
Department of Chemistry, Texas A&M University, College Station, Texas 77843, USA.
Raushel
F M
FM
D001665:Binding Sites; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D005971:Glutamates / Q000378:metabolism; D006834:Hydrazines / Q000378:metabolism; D006877:Hydroxamic Acids / Q000378:metabolism; D019811:Hydroxylamine / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation
D005971:Glutamates; D006834:Hydrazines; D006877:Hydroxamic Acids; C050614:glutamate-gamma-hydroxamic acid; C029424:hydrazine; D019811:Hydroxylamine; D002222:Carbamoyl-Phosphate Synthase (Ammonia)