Recombinant Escherichia coli GMP reductase: Kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme-ligand binary complex formation

Leonardo Krás Borges Martinelli, Rodrigo G. Ducati, Leonardo Astolfi Rosado, Ardala Breda, Bruna Pelegrim Selbach, Diógenes Santiago Santos, Luiz Augusto Basso

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Guanosine monophosphate (GMP) reductase catalyzes the reductive deamination of GMP to inosine monophosphate (IMP). GMP reductase plays an important role in the conversion of nucleoside and nucleotide derivatives of guanine to adenine nucleotides. In addition, as a member of the purine salvage pathway, it also participates in the reutilization of free intracellular bases. Here we present cloning, expression and purification of Escherichia coli guaC-encoded GMP reductase to determine its kinetic mechanism, as well as chemical and thermodynamic features of this reaction. Initial velocity studies and isothermal titration calorimetry demonstrated that GMP reductase follows an ordered bi-bi kinetic mechanism, in which GMP binds first to the enzyme followed by NADPH binding, and NADP+ dissociates first followed by IMP release. The isothermal titration calorimetry also showed that GMP and IMP binding are thermodynamically favorable processes. The pH-rate profiles showed groups with apparent pK values of 6.6 and 9.6 involved in catalysis, and pK values of 7.1 and 8.6 important to GMP binding, and a pK value of 6.2 important for NADPH binding. Primary deuterium kinetic isotope effects demonstrated that hydride transfer contributes to the rate-limiting step, whereas solvent kinetic isotope effects arise from a single protonic site that plays a modest role in catalysis. Multiple isotope effects suggest that protonation and hydride transfer steps take place in the same transition state, lending support to a concerted mechanism. Pre-steady-state kinetic data suggest that product release does not contribute to the rate-limiting step of the reaction catalyzed by E. coli GMP reductase.

Original languageEnglish (US)
Pages (from-to)1289-1305
Number of pages17
JournalMolecular BioSystems
Volume7
Issue number4
DOIs
StatePublished - Apr 1 2011
Externally publishedYes

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GMP Reductase
Guanosine Monophosphate
Thermodynamics
Inosine Monophosphate
Escherichia coli
Ligands
NADP
Isotopes
Enzymes
Calorimetry
Catalysis
Deamination
Guanine Nucleotides
Adenine Nucleotides
Deuterium
Nucleosides
Organism Cloning

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology

Cite this

Martinelli, L. K. B., Ducati, R. G., Rosado, L. A., Breda, A., Selbach, B. P., Santos, D. S., & Basso, L. A. (2011). Recombinant Escherichia coli GMP reductase: Kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme-ligand binary complex formation. Molecular BioSystems, 7(4), 1289-1305. https://doi.org/10.1039/c0mb00245c

Recombinant Escherichia coli GMP reductase : Kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme-ligand binary complex formation. / Martinelli, Leonardo Krás Borges; Ducati, Rodrigo G.; Rosado, Leonardo Astolfi; Breda, Ardala; Selbach, Bruna Pelegrim; Santos, Diógenes Santiago; Basso, Luiz Augusto.

In: Molecular BioSystems, Vol. 7, No. 4, 01.04.2011, p. 1289-1305.

Research output: Contribution to journalArticle

Martinelli, Leonardo Krás Borges ; Ducati, Rodrigo G. ; Rosado, Leonardo Astolfi ; Breda, Ardala ; Selbach, Bruna Pelegrim ; Santos, Diógenes Santiago ; Basso, Luiz Augusto. / Recombinant Escherichia coli GMP reductase : Kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme-ligand binary complex formation. In: Molecular BioSystems. 2011 ; Vol. 7, No. 4. pp. 1289-1305.
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