Analytical and graphical examination of strong binding by half-of-sites in proteins: illustration with aspartate transcarbamylase.

In multiple binding of ligands to a protein, the binding sites may seem to behave as if they are partitioned equally between two modalities. This paper analyzes three different molecular situations in which two actual assemblages appear: (i) two classes of sites exist at the outset in the ligand-free macromolecule; (ii) all sites are initially identical but after half are occupied, the affinity of the residual ones is altered; (iii) all sites are initially identical but they interact in a pairwise manner. The contours of affinity profiles-graphs of normalized stoichiometric binding constants (iK(i)) versus stoichiometric step number i-are examined for each situation to provide a basis for discriminating among them. Proper procedures for evaluating the site binding constants are then described. To illustrate these procedures, published experimental data for two real systems, binding of substrate or modifier by the enzyme aspartate transcarbamylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2), are scrutinized and the meaning of the calculated binding parameters is examined. The results demonstrate concretely that site binding constants cannot be specified without assuming a particular molecular model, but the stoichiometric constants can be assigned unambiguously without regard to the type of behavior at the individual sites.