Temperature dependence of binding and catalysis for the Cdc25B phosphatase.
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 (DeltaH(++) 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 (DeltaH(++) of 12 kcal/mol) is enthalpically more favorable than for the small molecule substrate p-nitrophenyl phosphate (DeltaH(++) of 18 kcal/mol), yet entropically less favorable (TDeltaS(++) 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.
Sohn
Jungsan
J
Department of Biochemistry and Chemistry, Duke University Medical Center, Durham, NC 27710, USA.
Rudolph
Johannes
J
D019943:Amino Acid Substitution; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D020687:cdc25 Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D019925:Cyclin A; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D051357:Cyclin-Dependent Kinase 2; D020687:cdc25 Phosphatases