The transition state of nucleoside hydrolase from the trypanosome Crithidia fasciculata has been characterized by multiple Vmax/Km kinetic isotope effects with labeled inosine and adenosine as substrates. Nucleoside hydrolase catalyzes the hydrolysis of the N-glycosidic linkage of the commonly occurring purine and pyrimidine nucleosides, with Vmax/Km ranging over 2 orders of magnitude. The kinetic isotope effects for inosine were [1′-3H] = 1.150 ± 0.006, [2′-3H] = 1.161 ± 0.003, [1′-14C] = 1.044 ± 0.004, [9-15N] = 1.026 ± 0.004, [4′-3H] = 0.992 ± 0.003, and [5′-3H] = 1.051 ± 0.003. The magnitude of the kinetic isotope effects for inosine, an equivalent [1′-3H] kinetic isotope effect for the poor substrate adenosine, and the rapid equilibrium random kinetic mechanism [Parkin D. W., Horenstein, B. A., Abdulah, D. R., Estupiñán, B., … Schramm, V. L. (1991) J. Biol. Chem. (in press)] all indicate that the isotope effects are fully expressed. The kinetic and solvent deuterium isotope effects have been used to analyze the transition-state structure using bond energy bond order vibrational analysis. The transition state involves a protonated hypoxanthine leaving group with a C-N glycosidic bond elongated to approximately 2 Å. The ribose group contains substantial carbocationic character, unusually strong hyperconjugation of H2′, and a bond length of approximately 3 Å to the incoming oxygen nucleophile. The remote isotope effect (4′-3H and 5′-3H) and the results of transition-state calculations provide the most detailed description of the steric and bonding properties of an enzyme-stabilized transition state.
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