QUANTIFYING THE ALLOSTERIC PROPERTIES OF ESCHERICHIA-COLI CARBAMYL-PHOSPHATE SYNTHETASE - DETERMINATION OF THERMODYNAMIC LINKED-FUNCTION PARAMETERS IN AN ORDERED KINETIC MECHANISM

The effects of the allosteric ligands UMP, IMP, and ornithine on the partial reactions catalyzed by Escherichia coli carbamyl phosphate synthetase have been examined. Both of these reactions, a HCO3--dependent ATP synthesis reaction and a carbamyl phosphate-dependent ATP synthesis reaction, follow bimolecular ordered sequential kinetic mechanisms. In the ATPase reaction, MgATP binds before HCO3- as established previously for the overall reaction catalyzed by carbamyl phosphate synthetase [Raushel, F. M., Anderson, P. M., & Villafranca, J. J. (1978) Biochemistry 17,5587-5591]. The initial velocity kinetics for the ATP synthesis reaction indicate that MgADP binds before carbamyl phosphate in an equilibrium ordered mechanism except in the presence of ornithine. Determination of true thermodynamic linked-function parameters describing the impact of allosteric ligands on the binding interactions of the first substrate to bind in an ordered mechanism requires experiments to be performed in which both substrates are varied even if only one is apparently affected by the allosteric ligands. In so doing, we have found that IMP has little effect on the overall reaction of either of these two partial reactions. UMP and ornithine, which have a pronounced effect on the apparent K(m) for MgATP in the overall reaction, both substantially change the thermodynamic dissociation constant for MgADP from the binary E-MgADP complex, K(ia), in the ATP synthesis reaction, with UMP increasing K(ia) 15-fold and ornithine decreasing K(ia) by 18-fold. By contrast, only UMP substantially affects the K(ia) for MgATP in the ATPase reaction, increasing it by 5-fold. These effects are independent of the concentration of the unaffected substrate, carbamyl phosphate in the ATP synthesis reaction and HCO3- in the ATPase reaction, and do not reflect the influence that the allosteric ligands have on the K(m) for nucleotide in each case. The UMP effect on the ATPase reaction and the ornithine effect on the ATP synthesis reaction derive primarily from changes in the off-rate constant for the nucleotide.