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

Rodrigo G Ducati1, Diógenes S Santos, Luiz A Basso

  • 1Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil.

Archives of Biochemistry and Biophysics
|May 7, 2009
PubMed

Insights

Mycobacterium tuberculosis purine nucleoside phosphorylase (MtPNP) targets tuberculosis persistence. This enzyme specifically phosphorylates 6-oxopurine nucleosides, not adenosine, via an ordered mechanism.

Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Purine nucleoside phosphorylase (PNP) from Mycobacterium tuberculosis (MtPNP) is a potential drug target for tuberculosis treatment.
  • Understanding MtPNP's enzymatic mechanism is crucial for developing novel anti-tubercular therapies.

Purpose of the Study:

  • To elucidate the kinetic mechanism and substrate specificity of Mycobacterium tuberculosis purine nucleoside phosphorylase (MtPNP).
  • To identify key catalytic residues and understand the role of proton transfer in the reaction mechanism.

Main Methods:

  • Enzyme kinetics studies including initial velocity, product inhibition, and equilibrium binding assays.
  • pH-rate profiles, proton inventory, and solvent deuterium isotope effect experiments.
  • Pre-steady-state kinetic analysis.

Main Results:

  • MtPNP exhibits specificity for natural 6-oxopurine nucleosides and synthetic compounds, but not adenosine.
  • The enzyme catalyzes 2'-deoxyguanosine phosphorolysis via a steady-state ordered bi bi kinetic mechanism.
  • A general acid catalyst is essential for catalysis and substrate binding, while product release influences the rate-limiting step.

Conclusions:

  • The determined kinetic mechanism provides insights into MtPNP's function in Mycobacterium tuberculosis.
  • This study lays the groundwork for structure-based drug design targeting MtPNP to combat tuberculosis.

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