This can be observed for some of the unnatural amino acids incorporated GRPs (Supporting Information Table 1) whereby although the binding constant is in the micromolar range, the protein still detects glucose in the millimolar ranges.
If this also applies to the V394L enzyme and the binding constant is diminished, the accumulation of GS could be explained on the basis of a kinetic phenomenon, based on the decreased ability of V394L to cleave the less preferred substrate.
Second and more importantly, UP1 exhibits Tel-22 G-quadruplex structural recognition in that the binding constant is on the order of 10 for Tel-22-Na+, 10 for Tel-22-K+, and 10 for Tel-22ss.
The ionic strength and pH dependence of the binding constant is modeled using the Debye-Huckel theory to account for the electrostatic free energy of the interacting species.
It should be noted that the determination of binding constant is less accurate at the higher NaCl because saturation could not be achieved in the assay, but it is clear that binding is significantly weakened.
However, in the case of UMP, UDP-Glc, and Glc-1-P, in which the substrate is bound directly to the binding phosphate, the binding constant is significantly smaller.
If the phosphate has no substituents covalently bound, as in the case of PP i, the binding constant is large.
Furthermore, because of the constraints imposed by global analysis of the data, the specific binding constant is also resolved with reasonable precision [ Ksp = (2.0 ± 1.6) × 10 M–1].
Here the equilibrium binding constant is increased almost 36-fold, mainly because of the increased dissociation rate (koff F82D-R85AA)/ koff (F82D) ~ 9).
The computationally calculated free energy change ΔG0 for TMF-HSA binding is −6.1 kcal.mol−1, and the binding constant is KTMF 1.2×103 M−1.2×103
