31PNMR was used to follow the positional isotope exchange reactions catalyzed by carbamoyl-phosphate synthetase from Escherichia coli. In agreement with the data of Wimmer et al. [Wimmer, M. J., Rose, I. A., Powers, S. G., & Meister, A. (1979) J. Biol. Chem. 254, 1854] carbamoyl-phosphate synthetase was shown to catalyze the βγ-bridge:β-nonbridge positional oxygen exchange in [γ-18O]ATP. The ratio of micromoles of ATP exchanged to micromoles of ADP produced was 0.42-0.46 in the presence or absence of L-ornithine. There was no detectable enzyme-catalyzed exchange in the presence of l-glutamine which is consistent with the previously published steady-state kinetic mechanism [Raushel, F. M., Anderson, P. M., & Villafranca, J. J. (1978) Biochemistry 17, 5587], These positional isotope-exchange data along with our rapid-quench data [Raushel, F. M., & Villafranca, J. J. (1979) Biochemistry 18, 3424] permit us to calculate the rate constants for the partitioning of intermediates in the reaction. The scheme is where EA is the enzyme-MgATP-HCO3 Michaelis complex and EP is the enzyme-MgADP-carboxy phosphate complex, and the values for k3, k4, and k5 are 4.2 s-1, 0.10 s-1, and 0.21 s-1, respectively. In the partial back-reaction of carbamoylphosphate synthetase, the enzyme was shown to catalyze the bridgemonbridge oxygen exchange in 18O-labeled carbamoyl phosphate in the presence of MgADP. The rate of exchange was 4 times faster than the net synthesis of ATP. This exchange reaction is consistent with the intermediate formation of carbamate. There was no detectable exchange in the absence of MgADP. Overall, these data support the formation of two intermediates, viz., carboxy phosphate and carbamate, in the overall reaction catalyzed by carbamoyl-phosphate synthetase. Both intermediates are formed faster than or equal to the fastest step in the reaction. © 1980, American Chemical Society. All rights reserved.
- Oxygen Isotopes
- Magnetic Resonance Spectroscopy
- Radioisotope Dilution Technique
- Adenosine Triphosphate
- Carbamoyl-phosphate Synthase (glutamine-hydrolyzing)
- Escherichia Coli