Electrostatic characterization of enzyme complexes

Evaluation of the mechanism of catalysis of dihydrofolate reductase

William R. Cannon, Barbara Jane Garrison, Stephen Benkovic

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

The catalytic mechanism of dihydrofolate reductase is evaluated with Poisson-Boltzmann electrostatic and quantum chemical vibrational frequency calculations. The results indicate that an elevated pK(a) of 6.5 associated with the chemical step is due to the formation of the enol tautomer of the substrate's pterin ring. The tautomer is induced to form as a result of substrate binding, in which the substrate desolvates the active site and binds to the carboxylate of Asp 27. Although the binding reaction is favorable, burial of the negative charge on Asp 27 is not. Protonation of Asp 27 occurs, concerted with tautomerization of the substrate, resulting in active site electrical neutrality and activation of the substrate for catalysis. These results require a reinterpretation of previous data from Raman spectroscopy studies in which it was proposed that the reactive atom, the pterin N5, is directly protonated. Quantum chemical vibrational frequency calculations demonstrate that the enol tautomer undergoes a Raman active vibrational perturbation at a frequency similar to that observed experimentally. Furthermore, the calculations indicate that direct protonation of the pterin N5 due to classical electrostatic interactions is quite difficult, with the pK(a) for this residue being shifted from 2.6 in solution to below zero while bound to the protein. The conclusions of this work are independent of the protein dielectric constant in the range of 4-20.

Original languageEnglish (US)
Pages (from-to)2386-2395
Number of pages10
JournalJournal of the American Chemical Society
Volume119
Issue number10
DOIs
StatePublished - Mar 12 1997

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Pterins
Tetrahydrofolate Dehydrogenase
Static Electricity
Catalysis
Electrostatics
Enzymes
Catalytic Domain
Substrates
Protonation
Burial
Vibrational spectra
Raman Spectrum Analysis
Proteins
Coulomb interactions
Raman spectroscopy
Permittivity
Chemical activation
Oxidoreductases
Atoms

All Science Journal Classification (ASJC) codes

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

Cite this

Cannon, William R. ; Garrison, Barbara Jane ; Benkovic, Stephen. / Electrostatic characterization of enzyme complexes : Evaluation of the mechanism of catalysis of dihydrofolate reductase. In: Journal of the American Chemical Society. 1997 ; Vol. 119, No. 10. pp. 2386-2395.
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abstract = "The catalytic mechanism of dihydrofolate reductase is evaluated with Poisson-Boltzmann electrostatic and quantum chemical vibrational frequency calculations. The results indicate that an elevated pK(a) of 6.5 associated with the chemical step is due to the formation of the enol tautomer of the substrate's pterin ring. The tautomer is induced to form as a result of substrate binding, in which the substrate desolvates the active site and binds to the carboxylate of Asp 27. Although the binding reaction is favorable, burial of the negative charge on Asp 27 is not. Protonation of Asp 27 occurs, concerted with tautomerization of the substrate, resulting in active site electrical neutrality and activation of the substrate for catalysis. These results require a reinterpretation of previous data from Raman spectroscopy studies in which it was proposed that the reactive atom, the pterin N5, is directly protonated. Quantum chemical vibrational frequency calculations demonstrate that the enol tautomer undergoes a Raman active vibrational perturbation at a frequency similar to that observed experimentally. Furthermore, the calculations indicate that direct protonation of the pterin N5 due to classical electrostatic interactions is quite difficult, with the pK(a) for this residue being shifted from 2.6 in solution to below zero while bound to the protein. The conclusions of this work are independent of the protein dielectric constant in the range of 4-20.",
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Electrostatic characterization of enzyme complexes : Evaluation of the mechanism of catalysis of dihydrofolate reductase. / Cannon, William R.; Garrison, Barbara Jane; Benkovic, Stephen.

In: Journal of the American Chemical Society, Vol. 119, No. 10, 12.03.1997, p. 2386-2395.

Research output: Contribution to journalArticle

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AB - The catalytic mechanism of dihydrofolate reductase is evaluated with Poisson-Boltzmann electrostatic and quantum chemical vibrational frequency calculations. The results indicate that an elevated pK(a) of 6.5 associated with the chemical step is due to the formation of the enol tautomer of the substrate's pterin ring. The tautomer is induced to form as a result of substrate binding, in which the substrate desolvates the active site and binds to the carboxylate of Asp 27. Although the binding reaction is favorable, burial of the negative charge on Asp 27 is not. Protonation of Asp 27 occurs, concerted with tautomerization of the substrate, resulting in active site electrical neutrality and activation of the substrate for catalysis. These results require a reinterpretation of previous data from Raman spectroscopy studies in which it was proposed that the reactive atom, the pterin N5, is directly protonated. Quantum chemical vibrational frequency calculations demonstrate that the enol tautomer undergoes a Raman active vibrational perturbation at a frequency similar to that observed experimentally. Furthermore, the calculations indicate that direct protonation of the pterin N5 due to classical electrostatic interactions is quite difficult, with the pK(a) for this residue being shifted from 2.6 in solution to below zero while bound to the protein. The conclusions of this work are independent of the protein dielectric constant in the range of 4-20.

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