Leaving group activation and pyrophosphate ionic state at the catalytic site of plasmodium falciparum orotate phosphoribosyltransferase

Yong Zhang, Hua Deng, Vern L. Schramm

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10 Citations (Scopus)

Abstract

Plasmodium falciparum orotate phosphoribosyltransferase (PfOPRT) catalyzes the reversible pyrophosphorolysis of orotidine 5′-monophosphate (OMP). Transition-state analysis from kinetic isotope effects supports a dianionic orotic acid (OA) leaving group. Isotope-edited Fourier transform infrared (FTIR) spectrometry complemented by homology modeling and quantum chemical calculations were used to characterize the orotate hydrogen-bond network for PfOPRT. Bond stretch frequencies for C2=O and C4=O of OMP were established from 13C-edited FTIR difference spectra. Both frequencies were shifted downward by 20 cm-1 upon formation of the Michaelis complex. Hydrogen-bond interactions to the orotate moiety induce strong leaving group polarization by ground-state destabilization. The C 2=O bond is 2.7 Å from two conserved water molecules, and the C4=O bond is within 2.4 Å of the NH2(ω) of Arg241 and the peptide NH of Phe97. Relative to free OMP, the N1 atom of PfOPRT-bound OMP indicates a ΔpKa of -4.6. The decreased basicity of N1 supports leaving group activation through a hydrogen-bond network at the PfOPRT active site. PfOPRT in complex with 18O-PPi and a proposed transition-state analogue revealed a trianionic PPi nucleophile with no significant P••O bond polarization, supporting a mechanism proceeding through the migration of the ribocation toward the PPi. These results along with previous PfOPRT transition-state analyses provide reaction coordinate information for the PfOPRT-catalyzed OMP pyrophosphorolysis reaction.

Original languageEnglish (US)
Pages (from-to)17023-17031
Number of pages9
JournalJournal of the American Chemical Society
Volume132
Issue number47
DOIs
StatePublished - Dec 1 2010

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Orotate Phosphoribosyltransferase
Plasmodium falciparum
Catalytic Domain
Hydrogen bonds
Chemical activation
Isotopes
Fourier transforms
Polarization
Infrared radiation
Nucleophiles
Hydrogen
Alkalinity
Ground state
Spectrometry
Peptides
Fourier Analysis
Atoms
Molecules
Kinetics
Orotic Acid

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

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title = "Leaving group activation and pyrophosphate ionic state at the catalytic site of plasmodium falciparum orotate phosphoribosyltransferase",
abstract = "Plasmodium falciparum orotate phosphoribosyltransferase (PfOPRT) catalyzes the reversible pyrophosphorolysis of orotidine 5′-monophosphate (OMP). Transition-state analysis from kinetic isotope effects supports a dianionic orotic acid (OA) leaving group. Isotope-edited Fourier transform infrared (FTIR) spectrometry complemented by homology modeling and quantum chemical calculations were used to characterize the orotate hydrogen-bond network for PfOPRT. Bond stretch frequencies for C2=O and C4=O of OMP were established from 13C-edited FTIR difference spectra. Both frequencies were shifted downward by 20 cm-1 upon formation of the Michaelis complex. Hydrogen-bond interactions to the orotate moiety induce strong leaving group polarization by ground-state destabilization. The C 2=O bond is 2.7 {\AA} from two conserved water molecules, and the C4=O bond is within 2.4 {\AA} of the NH2(ω) of Arg241 and the peptide NH of Phe97. Relative to free OMP, the N1 atom of PfOPRT-bound OMP indicates a ΔpKa of -4.6. The decreased basicity of N1 supports leaving group activation through a hydrogen-bond network at the PfOPRT active site. PfOPRT in complex with 18O-PPi and a proposed transition-state analogue revealed a trianionic PPi nucleophile with no significant P••O bond polarization, supporting a mechanism proceeding through the migration of the ribocation toward the PPi. These results along with previous PfOPRT transition-state analyses provide reaction coordinate information for the PfOPRT-catalyzed OMP pyrophosphorolysis reaction.",
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T1 - Leaving group activation and pyrophosphate ionic state at the catalytic site of plasmodium falciparum orotate phosphoribosyltransferase

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AU - Deng, Hua

AU - Schramm, Vern L.

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N2 - Plasmodium falciparum orotate phosphoribosyltransferase (PfOPRT) catalyzes the reversible pyrophosphorolysis of orotidine 5′-monophosphate (OMP). Transition-state analysis from kinetic isotope effects supports a dianionic orotic acid (OA) leaving group. Isotope-edited Fourier transform infrared (FTIR) spectrometry complemented by homology modeling and quantum chemical calculations were used to characterize the orotate hydrogen-bond network for PfOPRT. Bond stretch frequencies for C2=O and C4=O of OMP were established from 13C-edited FTIR difference spectra. Both frequencies were shifted downward by 20 cm-1 upon formation of the Michaelis complex. Hydrogen-bond interactions to the orotate moiety induce strong leaving group polarization by ground-state destabilization. The C 2=O bond is 2.7 Å from two conserved water molecules, and the C4=O bond is within 2.4 Å of the NH2(ω) of Arg241 and the peptide NH of Phe97. Relative to free OMP, the N1 atom of PfOPRT-bound OMP indicates a ΔpKa of -4.6. The decreased basicity of N1 supports leaving group activation through a hydrogen-bond network at the PfOPRT active site. PfOPRT in complex with 18O-PPi and a proposed transition-state analogue revealed a trianionic PPi nucleophile with no significant P••O bond polarization, supporting a mechanism proceeding through the migration of the ribocation toward the PPi. These results along with previous PfOPRT transition-state analyses provide reaction coordinate information for the PfOPRT-catalyzed OMP pyrophosphorolysis reaction.

AB - Plasmodium falciparum orotate phosphoribosyltransferase (PfOPRT) catalyzes the reversible pyrophosphorolysis of orotidine 5′-monophosphate (OMP). Transition-state analysis from kinetic isotope effects supports a dianionic orotic acid (OA) leaving group. Isotope-edited Fourier transform infrared (FTIR) spectrometry complemented by homology modeling and quantum chemical calculations were used to characterize the orotate hydrogen-bond network for PfOPRT. Bond stretch frequencies for C2=O and C4=O of OMP were established from 13C-edited FTIR difference spectra. Both frequencies were shifted downward by 20 cm-1 upon formation of the Michaelis complex. Hydrogen-bond interactions to the orotate moiety induce strong leaving group polarization by ground-state destabilization. The C 2=O bond is 2.7 Å from two conserved water molecules, and the C4=O bond is within 2.4 Å of the NH2(ω) of Arg241 and the peptide NH of Phe97. Relative to free OMP, the N1 atom of PfOPRT-bound OMP indicates a ΔpKa of -4.6. The decreased basicity of N1 supports leaving group activation through a hydrogen-bond network at the PfOPRT active site. PfOPRT in complex with 18O-PPi and a proposed transition-state analogue revealed a trianionic PPi nucleophile with no significant P••O bond polarization, supporting a mechanism proceeding through the migration of the ribocation toward the PPi. These results along with previous PfOPRT transition-state analyses provide reaction coordinate information for the PfOPRT-catalyzed OMP pyrophosphorolysis reaction.

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