A STRINGENT TEST FOR THE NUCLEOTIDE SWITCH MECHANISM OF CARBAMOYL-PHOSPHATE SYNTHETASE

Carbamoyl phosphate synthetase (CPS) catalyzes the formation of carbam oyl phosphate from bicarbonate, glutamine, and two molecules of MgATP. The X-ray crystal structure of the enzyme has revealed that the two n ucleotide binding sites are separated by approximately 35 Angstrom. Is otopic oxygen exchange of O-18 and O-16 between solvent water and [C-1 3]bicarbonate was measured using C-13 NMR spectroscopy during substrat e turnover in the presence and absence of glutamine as a nitrogen sour ce. In the absence of added glutamine, CPS catalyzed the exchange of o ne oxygen atom from bicarbonate with solvent water during every turnov er of the bicarbonate-dependent ATPase reaction. In the presence of ad ded glutamine, there was no exchange of solvent water with bicarbonate during the enzymatic synthesis of carbamoyl phosphate, indicating tha t any carbon-containing intermediate in the reaction mechanism is comm itted to the formation of carbamoyl phosphate and is not subject to hy drolysis. These results are fully consistent with a chemical mechanism that requires the physical migration of the carbamate intermediate fr om the site of its formation within one of the nucleotide binding doma ins to the other nucleotide binding domain for subsequent phosphorylat ion by the second MgATP. These results are not compatible with a nucle otide switch mechanism. The nucleotide switch mechanism includes the s ynthesis of carbamoyl phosphate entirely within a single nucleotide bi nding domain and concurrent conformational changes driven by the bicar bonate-dependent hydrolysis of MgATP at the second nucleotide binding domain.