title: The free radical of pyruvate formate-lyase. Characterization by EPR spectroscopy and involvement in catalysis as studied with the substrate-analogue hypophosphite.
abstract: The first-derivative EPR spectrum of the active form of Escherichia coli pyruvate formate-lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g = 2.0037). Isotope substitution studies demonstrated couplings of a carbon-centered unpaired electron to a solvent-exchangeable proton (a = 1.5 mT) and to further hydrogen nuclei (a = 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H-labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of 1 spin per active site. The formate analogue hypophosphite has been characterized as a specific kcat inhibitor of pyruvate formate-lyase which destroys the enzyme radical. Protein-linked 1-hydroxyethylphosphonate was previously described as the dead-end product after reaction of the analogue with the intermediary acetyl-enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178, 445-450]. EPR spectroscopy of this system has now identified the corresponding alpha-phosphoryl radical as a reaction intermediate [g = 2.0032; a(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half-life of about 20 min at 0 degrees C. This finding proves that the enzyme radical is a hydrogen-atom-transferring coenzymic element.
authors.affiliation: Institut für Biologische Chemie, Universität Heidelberg, Federal Republic of Germany.
authors.forename: V
authors.initials: V
authors.lastname: Unkrig
authors.forename: F A
authors.initials: FA
authors.lastname: Neugebauer
authors.forename: J
authors.initials: J
authors.lastname: Knappe
mesh_terms: D000123:Acetyltransferases / Q000037:antagonists & inhibitors; D004578:Electron Spin Resonance Spectroscopy; D005561:Formates / Q000378:metabolism; D005609:Free Radicals
chemical_list: D005561:Formates; D005609:Free Radicals; D000123:Acetyltransferases; C022560:formate C-acetyltransferase