Site-bound water and the shortcomings of a less than perfect transition state analogue

Kinetic measurements have shown that substantial enthalpy changes accompany substrate binding by cytidine deaminase, increasing markedly as the reacti on proceeds from the ground state (1/ K-m, DeltaH = - 13 kcal/mol) to the t ransition state (1/K-tx, DeltaH = -20 kcal/mol) [Snider, M. J., et al. (200 0) Biochemistry 39, 9746-9753]. In the present work, we determined the ther modynamic changes associated with the equilibrium binding of inhibitors by cytidine deaminase by isothermal titration calorimetry and van't Hoff analy sis of the temperature dependence of their inhibition constants. The result s indicate that the binding of the transition state analogue 3,4-dihydrouri dine DeltaH = -21 kcal/mol), like that of the transition state itself (Delt aH = -20 kcal/mol), is associated with a large favorable change in enthalpy . The significantly smaller enthalpy change that accompanies the binding of 3,4-dihydrozebularine (DeltaH = -10 kcal/mol), an analogue of 3,4-dihydrou ridine in which a hydrogen atom replaces this inhibitor's 4-OH group, is co nsistent with the view that polar interactions with the substrate at the si te of its chemical transformation play a critical role in reducing the enth alpy of activation for substrate hydrolysis. The entropic shortcomings of 3 ,4-dihydrouridine, in capturing all of the free energy involved in binding the actual transition state, may arise from its inability to displace a wat er molecule that occupies the binding site normally occupied by product amm onia.