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 journalArticle

20 Citations (Scopus)

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
Externally publishedYes

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Purine-Nucleoside Phosphorylase
Deoxyguanosine
Substrate Specificity
Mycobacterium tuberculosis
Kinetics
Purinones
Protons
Substrates
Deprotonation
Proton transfer
Deuterium
Guanine
Catalysis
Nucleosides
Isotopes
Adenosine
Tuberculosis
Phosphates
Equipment and Supplies
Acids

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Substrate specificity and kinetic mechanism of purine nucleoside phosphorylase from Mycobacterium tuberculosis. / Ducati, Rodrigo G.; Santos, Diógenes S.; Basso, Luiz A.

In: Archives of Biochemistry and Biophysics, Vol. 486, No. 2, 15.06.2009, p. 155-164.

Research output: Contribution to journalArticle

Ducati, Rodrigo G. ; Santos, Diógenes S. ; Basso, Luiz A. / Substrate specificity and kinetic mechanism of purine nucleoside phosphorylase from Mycobacterium tuberculosis. In: Archives of Biochemistry and Biophysics. 2009 ; Vol. 486, No. 2. pp. 155-164.
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