Interactive binding between the substrate and allosteric sites of carbamoyl-phosphate synthetase

Abstract

The interaction between Escherichia coli carbamoyl-phosphate synthetase (CPS) and a fluorescent analogue of an allosteric effector molecule, 1,N6-ethenoadenosine 5''-monophosphate (.epsilon.-AMP), has been detected by using fluorescence techniques and kinetic measurements. From fluorescence anisotropy titrations, it was found that .epsilon.-AMP binds to a single site on CPS with Kd = 0.033 mM. The nucleotide had a small activating effect on the rate of synthesis of carbamoyl phosphate but had no effect on the Km for ATP. To test whether .epsilon.-AMP binds to an allosteric site, allosteric effectors (UMP, IMP, and CMP), known to bind at the UMP/IMP site, were added to solutions containing the .epsilon.-AMP-CPS complex. With addition of these effector molecules, a progressive decrease of the fluorescence anisotropy was observed, indicating that bound .epsilon.-AMP was displaced by the allosteric effectors examined. From these titrations, the dissociation constants for UMP, IMP, CMP, ribose 5-phosphate, 2-doxyribose 5-phosphate, and orthophosphate were determined. When MgATP, a substrate, was employed as a titrant, the observed decrease in anisotropy was consistent with the formation of a ternary complex (.epsilon.-AMP-CPS-MgATP). The effect of ATP binding, monitored at the allosteric site, was magnesium dependent, and free magnesium in solution was required to obtain a hyperbolic binding isotherm. Solvent accessibility of .epsilon.-AMP in binary (.epsilon.-AMP-CPS) and ternary (.epsilon.-AMP-CPS-MgATP) complexes was determined from acrylamide quenching, showing that the base of .epsilon.-AMP is well shielded from the solvent even in the presence of MgATP. Using the theory of ligand binding [Weber, G. (1975) Adv. Protein Chem. 29, 1-83], we computed the free energy of coupling between MgATP and .epsilon.-AMP to be +0.48 kcal/mol. Thus, the experiments described in this paper provide evidence for antagonism (anticooperative interaction) between the substrate and allosteric sites and suggest that this interaction provides a method of regulating the physiological activity of CPS.