Carbamoyl phosphate synthetase: caught in the act of glutamine hydrolysis.
Carbamoyl phosphate synthetase from Escherichia coli catalyzes the production of carbamoyl phosphate from two molecules of Mg2+ATP, one molecule of bicarbonate, and one molecule of glutamine. The enzyme consists of two polypeptide chains referred to as the large and small subunits. While the large subunit provides the active sites responsible for the binding of nucleotides and other effector ligands, the small subunit contains those amino acid residues that catalyze the hydrolysis of glutamine to glutamate and ammonia. From both amino acid sequence analyses and structural studies it is now known that the small subunit belongs to the class I amidotransferase family of enzymes. Numerous biochemical studies have suggested that the reaction mechanism of the small subunit proceeds through the formation of the glutamyl thioester intermediate and that both Cys 269 and His 353 are critical for catalysis. Here we describe the X-ray crystallographic structure of carbamoyl phosphate synthetase from E. coli in which His 353 has been replaced with an asparagine residue. Crystals employed in the investigation were grown in the presence of glutamine, and the model has been refined to a crystallographic R-factor of 19.1% for all measured X-ray data from 30 to 1.8 A resolution. The active site of the small subunit clearly contains a covalently bound thioester intermediate at Cys 269, and indeed, this investigation provides the first direct structural observation of an enzyme intermediate in the amidotransferase family.
Thoden
J B
JB
Institute for Enzyme Research, The Graduate School, University of Wisconsin-Madison 53705, USA.
Miran
S G
SG
Phillips
J C
JC
Howard
A J
AJ
Raushel
F M
FM
Holden
H M
HM
D001216:Asparagine; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry* / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D005973:Glutamine / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D005973:Glutamine; D006639:Histidine; D001216:Asparagine; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)