Nucleoside Hydrolase From Crithidia Fasciculata: Metabolic Role, Purification, Specificity, and Kinetic Mechanism

Document Type

Article

Publication Date

1-1-1991

Description

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 x 103 M-1 s-1 to 1.7 x 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 given 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.

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