BINDING MODES FOR SUBSTRATE AND A PROPOSED TRANSITION-STATE ANALOG OFPROTOZOAN NUCLEOSIDE HYDROLASE

The transition-state structure for inosine-uridine nucleoside hydrolas e (IU-nucleoside hydrolase) from Crithidia fasciculata is characterize d by oxycarbonium character in the ribosyl and weak bonds to the depar ting hypoxanthine and incipient water nucleophile [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795]. Inhibitors designed to resemble the transition state ar e slow-onset, tight-binding inhibitors with observed K-m/K-i values up to 2 x 10(5) [Schramm, V. L., Horenstein, B. H., & Kline, P. C. (1994 ) J. Biol. Chem. 269, 18259-18262]. Although slow-onset, tight binding is consistent with transition-state stabilization, more direct eviden ce can be obtained by comparing the groups which interact with the sub strate to provide binding and catalysis with those which interact with the putative transition-state inhibitor. The K,value for inosine bind ing 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 n ear 8.0. A single group with pK of 9.1 must be protonated for catalyti c 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 (ph enyliminoribitol) 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 prof ile with the protonation of a single group, pK 7.5, required for inhib itor binding and the protonation of a subsequent group, pK 6.6, causin g loss of binding. It has been proposed that the positive charge of pr otonated inhibitor (pK 6.5) is a recognition feature for binding as a transition-state inhibitor. However, the pH analysis indicates that th e neutral inhibitor is the preferred species for binding the active fo rm of the enzyme. The slow-onset phase of phenyliminoribitol binding d isappears at low pH, suggesting that a time-dependent protonation of t he bound complex could be responsible for the slow-onset phase of inhi bition.