Regulatory control of the amidotransferase domain of carbamoyl phosphate synthetase.
Carbamoyl phosphate synthetase catalyzes the hydrolysis of glutamine by the nucleophilic attack of an active site cysteine residue through a mechanism that requires the formation of a gamma-glutamyl thioester intermediate. The steady-state mole fraction of the thioester intermediate was determined to be 0.23 in the presence and absence of ATP and bicarbonate. The kinetics of formation and hydrolysis of the gamma-glutamyl thioester intermediate during CPS catalyzed hydrolysis of glutamine were determined. When ATP and bicarbonate are added to CPS and glutamine, the kcat for glutamine hydrolysis increases from 0.17 to 150 min-1. The observed rate constant for thioester intermediate formation increases from 18 to 580 min-1, and the microscopic rate constant for hydrolysis of the intermediate increases from 0.15 to 460 min-1. These results demonstrate the kinetic competence of the thioester intermediate during glutamine hydrolysis. The rate-determining step changes from the hydrolysis of the intermediate when ATP and bicarbonate are absent to the formation of the intermediate upon the addition of ATP and bicarbonate. The 3 order of magnitude increase in the rate of glutamine hydrolysis upon the addition of ATP and bicarbonate is indicative of the allosteric communication between two of the three reaction centers of CPS. These sites are physically separated by approximately 45 A.
Miles
B W
BW
Department of Chemistry, Texas A&M University, College Station 77843, USA.
Banzon
J A
JA
Raushel
F M
FM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000878:Anthranilate Synthase; D001639:Bicarbonates / Q000378:metabolism; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D019884:Nitrogenous Group Transferases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D001639:Bicarbonates; D004151:Dipeptides; D000255:Adenosine Triphosphate; D019884:Nitrogenous Group Transferases; D000878:Anthranilate Synthase; C020655:anthranilate synthase, glutamine amidotransferase subunit; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D005978:Glutathione; C017341:gamma-glutamylcysteine