Transition-State Analysis of Nucleoside Hydrorlase from Crithidia fasciculata

Benjamin A. Horenstein, David W. Parkin, Vern L. Schramm, Vern L. Schramm

Research output: Contribution to journalArticle

150 Citations (Scopus)

Abstract

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 Kmax/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 A. 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.

Original languageEnglish (US)
Pages (from-to)10788-10795
Number of pages8
JournalBiochemistry
Volume30
Issue number44
StatePublished - 1991

Fingerprint

Crithidia fasciculata
Nucleosides
Isotopes
Inosine
Kinetics
N-Glycosyl Hydrolases
Adenosine
Pyrimidine Nucleosides
Purine Nucleosides
Nucleophiles
Hypoxanthine
Ribose
Trypanosomiasis
Deuterium
Bond length
Substrates
Hydrolysis
Oxygen

ASJC Scopus subject areas

  • Biochemistry

Cite this

Horenstein, B. A., Parkin, D. W., Schramm, V. L., & Schramm, V. L. (1991). Transition-State Analysis of Nucleoside Hydrorlase from Crithidia fasciculata. Biochemistry, 30(44), 10788-10795.

Transition-State Analysis of Nucleoside Hydrorlase from Crithidia fasciculata. / Horenstein, Benjamin A.; Parkin, David W.; Schramm, Vern L.; Schramm, Vern L.

In: Biochemistry, Vol. 30, No. 44, 1991, p. 10788-10795.

Research output: Contribution to journalArticle

Horenstein, BA, Parkin, DW, Schramm, VL & Schramm, VL 1991, 'Transition-State Analysis of Nucleoside Hydrorlase from Crithidia fasciculata', Biochemistry, vol. 30, no. 44, pp. 10788-10795.
Horenstein, Benjamin A. ; Parkin, David W. ; Schramm, Vern L. ; Schramm, Vern L. / Transition-State Analysis of Nucleoside Hydrorlase from Crithidia fasciculata. In: Biochemistry. 1991 ; Vol. 30, No. 44. pp. 10788-10795.
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