Temperature dependence of binding and catalysis for the Cdc25B phosphatase Original Research Article

Using a combination of steady-state and single-turnover kinetics, we probe the temperature dependence of substrate association and chemistry for the reaction of Cdc25B phosphatase with its Cdk2–pTpY/CycA protein substrate. The transition state for substrate association is dominated by an enthalpic barrier (ΔH‡ of 13 kcal/mol) and has a favorable entropic contribution of 4 kcal/mol at 298 K. Phosphate transfer from Cdk2–pTpY/CycA to enzyme (ΔH‡ of 12 kcal/mol) is enthalpically more favorable than for the small molecule substrate p-nitrophenyl phosphate (ΔH‡ of 18 kcal/mol), yet entropically less favorable (TΔS‡ of 2 vs. − 6 kcal/mol at 298 K, respectively). By measuring the temperature dependence of binding and catalysis for several hotspot mutants involved in binding of protein substrate, we determine the enthalpy–entropy compensations for changes in rates of association and phosphate transfer compared to the wild type system. We conclude that the transition state for enzyme–substrate association involves tight and specific contacts at the remote docking site and that phospho-transfer from Cdk2–pTpY/CycA to the pre-organized active site of the enzyme is accompanied by unfavorable entropic rearrangements that promote rapid product dissociation.