Kinetics of the gamma-glutamyl thioester intermediate in the glutamine amidotransferase activity of carbamoyl phosphate synthetase of E-coli

The chemical mechanism for the amidotransferase activity of carbamoyl phosphate synthetase is thought to involve the formation of a 7-glutamyl thioester intermediate. This covalent intermediate can be isolated and stabilized by acid precipitation. The steady-state concentration of the intermediate was determined to be 0.23 mole fraction of 7-glutamyl enzyme. The kinetics of formation and decay for this intermediate were determined both in the presence and absence of ATP and bicarbonate. With ATP and bicarbonate, the rate of intermediate formation was 150 min-1, and the rate of decay was 510 min-I. Both rates are consistent with the observed kcat for the overall reaction of 156 min-1. In the absence of ATP and bicarbonate, the observed rate of formation of the intermediate was 18 min-i, and the rate of decay was determined to be 0.15 min-I which is comparable to the observed steady state turnover of ghtamine, 0.27 rain-1. The addition of ATP and bicarbonate increases the overall rate of glutamine hydrolysis by a factor of 550 with a 10 fold increase in the rate of 7-glutamyl thioester formation and a 3400 fold enhancement for the rate of intermediate decay.

Kinetics of the gamma-glutamyl thioester intermediate in the glutamine amidotransferase activity of carbamoyl phosphate synthetase of E-coli Academic Article