Binding of allosteric effectors to carbamyl-phosphate synthetase from Escherichia coli

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Abstract
The binding of ornithine and IMP, positive allosteric effectors and of UMP, a negative allosteric effector, to carbamyl-phosphate synthetase from E. coli was studied by equilibrium dialysis. The monomeric form of the enzyme has 1 binding site for each of the 3 allosteric ligands. The binding of UMP is inhibited by ornithine, IMP, MgATP and NH3 (also a positive allosteric effector). Bicarbonate, L-glutamine and ATP (Mg2+ absent) had no effect on the binding of UMP. The affinity of the enzyme for UMP was increased if phosphate buffer was replaced by Tris buffer. The binding of ornithine was inhibited by UMP and NH3, enhanced by MgATP, MgADP and IMP, and not affected by bicarbonate, L-glutamine or ATP (Mg2+ absent). Ornithine and NH3 probably bind to the same site on the enzyme. The binding of IMP is facilitated by ornithine and NH3, but is inhibited by MgATP or ATP, indicating that adenine nucleotides can also bind to the IMP binding site. The results of these binding studies are consistent with a scheme previously proposed in which the allosteric effectors function by stabilizing 1 or the other of 2 different conformational states of the enzyme which are in equilibrium with each other. According to this scheme, binding of the substrate MgATP is greatly facilitated when the enzyme exists in the conformational state stabilized by the positive allosteric effectors.