Substrate specificity and kinetic mechanism of purine nucleoside phosphorylase from Mycobacterium tuberculosis

Rodrigo G. Ducati, Diógenes S. Santos, Luiz A. Basso

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

Purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) is numbered among targets for persistence of the causative agent of tuberculosis. Here, it is shown that MtPNP is more specific to natural 6-oxopurine nucleosides and synthetic compounds, and does not catalyze the phosphorolysis of adenosine. Initial velocity, product inhibition and equilibrium binding data suggest that MtPNP catalyzes 2′-deoxyguanosine (2dGuo) phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate (Pi) binds first followed by 2dGuo, and ribose 1-phosphate dissociates first followed by guanine. pH-rate profiles indicated a general acid as being essential for both catalysis and 2dGuo binding, and that deprotonation of a group abolishes Pi binding. Proton inventory and solvent deuterium isotope effects indicate that a single solvent proton transfer makes a modest contribution to the rate-limiting step. Pre-steady-state kinetic data indicate that product release appears to contribute to the rate-limiting step for MtPNP-catalyzed reaction.

Original languageEnglish (US)
Pages (from-to)155-164
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume486
Issue number2
DOIs
StatePublished - Jun 15 2009

Keywords

  • Enzyme kinetic mechanism
  • Fluorescence titration
  • Initial velocity
  • PNP
  • Pre-steady-state kinetics
  • Product inhibition
  • Purine nucleoside phosphorylase
  • Solvent isotope effects
  • Substrate specificity
  • pH-rate profiles

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'Substrate specificity and kinetic mechanism of purine nucleoside phosphorylase from Mycobacterium tuberculosis'. Together they form a unique fingerprint.

Cite this