Studies with substrate and cofactor analogues provide evidence for a radical mechanism in the chorismate synthase reaction

J Biol Chem. 2000 Nov 17;275(46):35825-30. doi: 10.1074/jbc.M005796200.

Abstract

Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The strict requirement for a reduced FMN cofactor and a trans-1,4-elimination are unusual. (6R)-6-Fluoro-EPSP was shown to be converted to chorismate stoichiometrically with enzyme-active sites in the presence of dithionite. This conversion was associated with the oxidation of FMN to give a stable flavin semiquinone. The IC(50) of the fluorinated substrate analogue was 0.5 and 250 microm with the Escherichia coli enzyme, depending on whether it was preincubated with the enzyme or not. The lack of dissociation of the flavin semiquinone and chorismate from the enzyme appears to be the basis of the essentially irreversible inhibition by this analogue. A dithionite-dependent transient formation of flavin semiquinone during turnover of (6S)-6-fluoro-EPSP has been observed. These reactions are best rationalized by radical chemistry that is strongly supportive of a radical mechanism occurring during normal turnover. The lack of activity with 5-deaza-FMN provides additional evidence for the role of flavin in catalysis by the E. coli enzyme.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzoquinones / chemistry
  • Benzoquinones / metabolism
  • Chromatography, High Pressure Liquid
  • Dithionite / pharmacology
  • Escherichia coli / enzymology*
  • Flavin Mononucleotide / analogs & derivatives
  • Flavin Mononucleotide / chemistry
  • Flavin Mononucleotide / metabolism*
  • Inhibitory Concentration 50
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Molecular Structure
  • Organophosphorus Compounds / chemistry
  • Organophosphorus Compounds / metabolism
  • Organophosphorus Compounds / pharmacology
  • Oxidation-Reduction
  • Phosphorus-Oxygen Lyases / antagonists & inhibitors
  • Phosphorus-Oxygen Lyases / chemistry
  • Phosphorus-Oxygen Lyases / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Shikimic Acid / analogs & derivatives
  • Shikimic Acid / chemistry
  • Shikimic Acid / metabolism
  • Shikimic Acid / pharmacology
  • Spectrophotometry

Substances

  • 6-fluoro-5-enolpyruvylshikimate-3-phosphate
  • Benzoquinones
  • Organophosphorus Compounds
  • Recombinant Proteins
  • Dithionite
  • 5-enolpyruvoylshikimate-3-phosphate
  • Shikimic Acid
  • semiquinone radicals
  • 5-deaza-FMN
  • Flavin Mononucleotide
  • chorismate synthase
  • Phosphorus-Oxygen Lyases