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

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14 Scopus citations


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
Issue number25
StatePublished - Jun 29 2011

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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