CARBAMOYL-PHOSPHATE SYNTHETASE - CAUGHT IN THE ACT OF GLUTAMINE HYDROLYSIS

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the pro duction of carbamoyl phosphate from two molecules of Mg(2+)ATP, one mo lecule of bicarbonate, and one molecule of glutamine. The enzyme consi sts of two polypeptide chains referred to as the large and small subun its. While the large subunit provides the active sites responsible for the binding of nucleotides and other effector ligands, the small subu nit contains those amino acid residues that catalyze the hydrolysis of glutamine to glutamate and ammonia. From both amino acid sequence ana lyses and structural studies it is now known that the small subunit be longs to the class I amidotransferase family of enzymes. Numerous bioc hemical studies have suggested that the reaction mechanism of the smal l subunit proceeds through the formation of the glutamyl thioester int ermediate and that both Cys 269 and His 353 are critical for catalysis . Here we describe the X-ray crystallographic structure of carbamoyl p hosphate synthetase from E, coli in which His 353 has been replaced wi th 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 Angstrom resolution. The active site of the small subunit cl early contains a covalently bound thioester intermediate at Cys 269, a nd indeed, this investigation provides the first direct structural obs ervation of an enzyme intermediate in the amidotransferase family.