Uridine phosphorylase from Trypanosoma cruzi: Kinetic and chemical mechanisms

Rafael G. Silva, Vern L. Schramm

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

The reversible phosphorolysis of uridine to generate uracil and ribose 1-phosphate is catalyzed by uridine phosphorylase and is involved in the pyrimidine salvage pathway. We define the reaction mechanism of uridine phosphorylase from Trypanosoma cruzi by steady-state and pre-steady-state kinetics, pH-rate profiles, kinetic isotope effects from uridine, and solvent deuterium isotope effects. Initial rate and product inhibition patterns suggest a steady-state random kinetic mechanism. Pre-steady-state kinetics indicated no rate-limiting step after formation of the enzyme-products ternary complex, as no burst in product formation is observed. The limiting single-turnover rate constant equals the steady-state turnover number; thus, chemistry is partially or fully rate limiting. Kinetic isotope effects with [1′- 3H]-, [1′- 14C]-, and [5′- 14C,1,3- 15N 2]uridine gave experimental values of α-T(V/K) uridine = 1.063, 14(V/K) uridine = 1.069, and 15,β-15(V/K) uridine = 1.018, in agreement with an A ND N (S N2) mechanism where chemistry contributes significantly to the overall rate-limiting step of the reaction. Density functional theory modeling of the reaction in gas phase supports an A ND N mechanism. Solvent deuterium kinetic isotope effects were unity, indicating that no kinetically significant proton transfer step is involved at the transition state. In this N-ribosyl transferase, proton transfer to neutralize the leaving group is not part of transition state formation, consistent with an enzyme-stabilized anionic uracil as the leaving group. Kinetic analysis as a function of pH indicates one protonated group essential for catalysis and for substrate binding.

Original languageEnglish (US)
Pages (from-to)9158-9166
Number of pages9
JournalBiochemistry
Volume50
Issue number42
DOIs
StatePublished - Oct 25 2011

ASJC Scopus subject areas

  • Biochemistry

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