Nucleoside hydrolase from Crithidia fasciculata: Metabolic role, purification, specificity, and kinetic mechanism

Crithidia fasciculata cells grown on complex medium with added [8-¹⁴C,5′-³H]inosine or [8-¹⁴C,5′-³H]adenosine metabolize >50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to >99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of M_r 34,000 subunits to give an apparent holoenzyme M_r of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The K_m values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of V_max/K_m vary from 3.4 × 10³ M^-1 s^-1 to 1.7 × 10⁵ M^-1 s^-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s^-1 at 30°C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental K_eq of 106 M with H₂O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5′-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pK_a values of 6.1 with decreasing V_max/K_m values below the pK_a and a plateau at higher pH values. These effects are due to the pH sensitivity of the V_max values, since K_m is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.