Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants

The binding of several phosphonodifluoromethyl phenylalanine (F(2)Pmp)-cont aining peptides to protein-tyrosine phosphatase 1B (PTP1B) and its substrat e-trapping mutants (C215S and D181A) has been studied using isothermal titr ation calorimetry. The binding of a high affinity ligand, Ac-Asp-Ala-Asp-Gl u-F(2)Pmp-Leu-NH2, to PTP1B (K-d = 0.24 muM) is favored by both enthalpic a nd entropic contributions. Disruption of ionic interactions between the sid e chain of Arg-47 and the N-terminal acidic residues reduces the binding af finity primarily through the reduction of the T DeltaS term. The role of Ar g-47 may be to maximize surface contact between PTP1B and the peptide, whic h contributes to high affinity binding. The active site Cys-215-->Ser mutan t PTP1B binds ligands with the same affinity as the wild-type enzyme. Howev er, unlike wild-type PTP1B, peptide binding to C215S is predominately drive n by enthalpy change, which likely results from the elimination of the elec trostatic repulsion between the thiolate anion and the phosphonate group. T he increased enthalpic contribution is offset by reduction in the binding e ntropy, which may be the result of increased entropy of the unbound protein caused by this mutation. The general acid-deficient mutant D181A binds the peptide 5-fold tighter than the C215S mutant, consistent with the observat ion that the Asp to Ala mutant is a better "substrate-trapping" reagent tha n C215S, The increased binding affinity for D181A as compared with the wild type PTP1B results primarily from an increase in the DeltaH of binding in t he mutant, which may be related to decreased electrostatic repulsion betwee n the phosphate moiety and PTP1B, These results have important implications for the design of high affinity PTP1B inhibitors.