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

David W. Parkin, Benjamin A. Horenstein, Dorina R. Abdulah, Bernardo Estupiñán, Vern L. Schramm

Research output: Contribution to journalArticle

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Abstract

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 Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km 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 Keq 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 pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.

Original languageEnglish (US)
Pages (from-to)20658-20665
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number31
StatePublished - Nov 5 1991
Externally publishedYes

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N-Glycosyl Hydrolases
Crithidia fasciculata
Inosine
Purification
Nucleosides
Kinetics
Substrates
Enzymes
Purine-Nucleoside Phosphorylase
Pyrimidine Nucleosides
Purine Nucleosides
Pyrimidines
Holoenzymes
Nucleophiles
Hypoxanthine
Ribose
Enzyme Inhibitors
Glycosides
Adenosine
Catalysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Nucleoside hydrolase from Crithidia fasciculata : Metabolic role, purification, specificity, and kinetic mechanism. / Parkin, David W.; Horenstein, Benjamin A.; Abdulah, Dorina R.; Estupiñán, Bernardo; Schramm, Vern L.

In: Journal of Biological Chemistry, Vol. 266, No. 31, 05.11.1991, p. 20658-20665.

Research output: Contribution to journalArticle

Parkin, David W. ; Horenstein, Benjamin A. ; Abdulah, Dorina R. ; Estupiñán, Bernardo ; Schramm, Vern L. / Nucleoside hydrolase from Crithidia fasciculata : Metabolic role, purification, specificity, and kinetic mechanism. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 31. pp. 20658-20665.
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AB - 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 Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km 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 Keq 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 pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.

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