Protein affinities for small molecules: conceptions and misconceptions.
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Binding Sites↗
Biomedical subjects
D L Hunston
Publications and source records attributed to D L Hunston.
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.
Aspartate Carbamoyltransferase↗
Protein interactions with small molecules. Relationships between stoichiometric binding constants, site binding constants, and empirical binding parameters.
The multiple equilibria for the binding of a ligand A by a macromolecule P with n binding sites may be formulated in terms of a stoichiometric analysis or on the basis of a site-oriented scrutiny. The dependence of binding on ligand concentration can always be correlated in terms of n stoichiometric binding constants,Ki, even if there are interactions between sites that accentuate or attenuate binding affinities. A corresponding correlation in terms of site binding constants, kj, under the most general circumstances depends on the definition of n2n-1 different constants of which 2n-1 are independent. If experimental data are correlated in terms of n parameters kalpha, kbeta ... klambda in an equation of the site-binding form, (see article for formular) then there is no guarantee that the values of ka, kb, etc., have any unique relationships to site binging constants. Examples are given to illustrate this point. Equation are derived for relating stoichiometric binding constants to site binding constants, for the general case and for various special circumstances. These equations make it possible to define and analyze binding insystems with interactions and conformational accommodations. Accordingly, a graphical procedure is described (in which iKi is plotted against i, the stoichiometric binding step) that provides an affinity profile for concise representation of magnitudes of binding constants and for detecting interactions that accentuate or attenuate site binding affinities.
Binding Sites↗