Low-affinity binding determined by titration calorimetry using a high-affinity coupling ligand: a thermodynamic study of ligand binding to protein tyrosine phosphatase 1B.

Abstract A competition-based method is used for the determination of the thermodynamic parameters for a low-affinity ligand binding reaction by isothermal titration calorimetry. This method is based on the coupling of a high-affinity ligand to the binding of the low-affinity ligand. Results are presented for the binding of a nonhydrolyzable phosphotyrosine analog phosphonodifluoromethyl phenylalanine (F 2 Pmp)-containing peptide (Ac-Asp-Ala-Asp-Glu-F 2 Pmp-Leu-NH 2 ), arsenate, and inorganic phosphate to the intracellular human protein tyrosine phosphatase 1B(PTP1B). The binding constants are 3.3 × 10 6 , 4.3 × 10 3 , and 48 M −1 for the F 2 Pmp-containing peptide, arsenate, and inorganic phosphate, respectively. The binding of arsenate and inorganic phosphate to PTP1B is enthalpy driven. This is in contrast to the binding of the F 2 Pmp-containing peptide which is mainly driven by entropy. The calorimetrically determined binding constants are in agreement with the K i values determined by enzyme inhibition studies. This demonstrates that isothermal titration calorimetry can be used to quantitatively determine the thermodynamic parameters for the interactions between proteins and low-affinity ligands if a proper coupling ligand can be identified.