Adenosine Deaminase: Solvent Isotope and pH Effects on the Binding of Transition-State and Ground-State Analogue Inhibitors

We have studied the pH(D) effects on the deamination of adenosine catalyzed by adenosine deaminase as well as on the binding of the inhibitors purine riboside, a ground-state analogue, and 1,6-dihydro-6-(hydroxymethyl)purine riboside (DHMPR), a transition-state analogue. The observed pK_a value of 4.9 for the free enzyme in H₂O buffers is found to be increased by 0.6 pK unit in the enzyme-substrate complex and decreased by 0.5–1.0 pK unit in the enzyme-inhibitor complexes. In D₂O buffers, the pK_as of the free enzyme and its complexes are all found to be increased by ∼0.6 pK unit relative to their position in H₂O. A small inverse solvent isotope effect is observed on V_max while none is observed on K_m. Substantial solvent isotope effects [K_i(H₂O)/K_i(D₂O) = 1.2–1.5] are found for the dissociation of both ground-state and transition-state analogue inhibitors from the enzyme-inhibitor complex. Fluorescence titrations of the enzyme with DHMPR in H₂O and D₂O confirm the equilibrium solvent isotope effect obtained from kinetic experiments. For the transition-state analogue, a small inverse kinetic effect, similar in magnitude to that on V_max, is found on the association rate constant, k_on, while a normal effect is observed on the dissociation rate constant, k_off. The intrinsic protein fluorescence is quenched 70% by the transition-state analogue and only 6% by the ground-state analogue supporting the idea that a greater structural reorganization of the enzyme is required to bind the transition state effectively in comparison to the ground state. In contrast, a large UV difference spectrum is observed upon formation of the complex with purine riboside, suggesting that the binding isotope effect may be interpretable in terms of structural changes in the ligand rather than in the enzyme. Three possible structures for the complexed inhibitors are discussed which could account for the observed solvent isotope effect. The data are most consistent with protonation of the purine ring at N-1 by an active-site sulfhydryl. However, hydration of the purine ring at C-6 or formation of a covalent sulfhydryl adduct at C-2 or C-6 cannot be excluded in view of the large pK_a shifts required to accomplish purine protonation with a sulfhydryl group.