Transition-state analysis of Trypanosoma cruzi uridine phosphorylase- catalyzed arsenolysis of uridine

Rafael G. Silva, Mathew J. Vetticatt, Emilio F. Merino, Maria B. Cassera, Vern L. Schramm

Research output: Contribution to journalArticle

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Abstract

Uridine phosphorylase catalyzes the reversible phosphorolysis of uridine and 2′-deoxyuridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli, purified to homogeneity, and shown to be a homodimeric uridine phosphorylase, with similar specificity for uridine and 2′-deoxyuridine and undetectable activity toward thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1′-14C = 1.103, 1,3-15N 2 = 1.034, 3-15N = 1.004, 1-15N = 1.030, 1′-3H = 1.132, 2′-2H = 1.086, and 5′-3H2 = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition-state structure and geometry. Matching of experimental KIEs to proposed transition-state structures suggests an almost synchronous, S N2-like transition-state model, in which the ribosyl moiety possesses significant bond order to both nucleophile and leaving groups. Natural bond orbital analysis allowed a comparison of the charge distribution pattern between the ground-state and the transition-state models.

Original languageEnglish (US)
Pages (from-to)9923-9931
Number of pages9
JournalJournal of the American Chemical Society
Volume133
Issue number25
DOIs
StatePublished - Jun 29 2011

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Uridine Phosphorylase
Trypanosoma cruzi
Uridine
Isotopes
Deoxyuridine
Nucleophiles
Kinetics
Purine Nucleosides
Salvaging
Uracil
Charge distribution
Electron transitions
Thymidine
Escherichia coli
Ground state
Density functional theory
Phosphates
Genes
Genome
Geometry

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Transition-state analysis of Trypanosoma cruzi uridine phosphorylase- catalyzed arsenolysis of uridine. / Silva, Rafael G.; Vetticatt, Mathew J.; Merino, Emilio F.; Cassera, Maria B.; Schramm, Vern L.

In: Journal of the American Chemical Society, Vol. 133, No. 25, 29.06.2011, p. 9923-9931.

Research output: Contribution to journalArticle

Silva, Rafael G. ; Vetticatt, Mathew J. ; Merino, Emilio F. ; Cassera, Maria B. ; Schramm, Vern L. / Transition-state analysis of Trypanosoma cruzi uridine phosphorylase- catalyzed arsenolysis of uridine. In: Journal of the American Chemical Society. 2011 ; Vol. 133, No. 25. pp. 9923-9931.
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