Binding Modes for Substrate and a Proposed Transition-State Analogue of Protozoan Nucleoside Hydrolase

The transition-state structure for inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase) from Crithidia fasciculata is characterized by oxycarbonium character in the ribosyl and weak bonds to the departing hypoxanthine and incipient water nucleophile [Horenstein, B. A., Parkin, D. W., Estupiñán, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795], Inhibitors designed to resemble the transition state are slow-onset, tight-binding inhibitors with observed K_m/K_i values up to 2 x 10⁵ [Schramm, V. L., Horenstein, B. H., & Kline, P. C. (1994) J. Biol. Chem. 269, 18259-18262], Although slowonset, tight binding is consistent with transition-state stabilization, more direct evidence can be obtained by comparing the groups which interact with the substrate to provide binding and catalysis with those which interact with the putative transition-state inhibitor. The K_m value for inosine binding to IU-nucleoside hydrolase is independent of pH over the range 5.6-10.5. Dependencies of V_max and V_max/K_m on pH result in pH optima near 8.0. A single group with pK of 9.1 must be protonated for catalytic activity, and protonation of a second group with a pK of 7.1 results in loss of activity. 1 -(S)-Phenyl-1,4-dideoxy-1,4- imino-D-ribitol (phenyliminoribitol) binds with an equilibrium K_d of 30 nM and has been proposed to be a transition-state inhibitor. The pH dependence for the competitive inhibition by phenyliminoribitol resembles the V_max profile with the protonation of a single group, pK 7.5, required for inhibitor binding and the protonation of a subsequent group, pK 6.6, causing loss of binding. It has been proposed that the positive charge of protonated inhibitor (pK 6.5) is a recognition feature for binding as a transition-state inhibitor. However, the pH analysis indicates that the neutral inhibitor is the preferred species for binding the active form of the enzyme. The slow-onset phase of phenyliminoribitol binding disappears at low pH, suggesting that a time-dependent protonation of the bound complex could be responsible for the slow-onset phase of inhibition.