The free radical in pyruvate formate-lyase is located on glycine-734.
Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherichia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure. By EPR spectroscopy of selectively 13C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central 13C nucleus (A parallel = 4.9 mT and A perpendicular = 0.1 mT) and to 13C nuclei in alpha and beta positions agree with literature data for glycine radical models. N-coupling was verified through uniform 15N-labeling. The large 1H hyperfine splitting (1.5 mT) dominating the EPR spectrum was assigned to the alpha proton, which in the enzyme radical is readily solvent-exchangeable. Oxygen destruction of the radical produced two unique fragments (82 and 3 kDa) of the constituent polypeptide chain. The N-terminal block on the small fragment was identified by mass spectrometry as an oxalyl residue that derives from Gly-734, thus assigning the primary structural glycyl radical position. The carbon-centered radical is probably resonance-stabilized through the adjacent carboxamide groups in the polypeptide main chain and could be comparable energetically with other known protein radicals carrying the unpaired electron in tyrosine or tryptophan residues.
Wagner
A F
AF
Institut für Biologische Chemie, Universität Heidelberg, Federal Republic of Germany.
Frey
M
M
Neugebauer
F A
FA
Schäfer
W
W
Knappe
J
J
D000123:Acetyltransferases / Q000737:chemistry*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D005609:Free Radicals; D005998:Glycine / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry
D001426:Bacterial Proteins; D005609:Free Radicals; D010446:Peptide Fragments; D000123:Acetyltransferases; C022560:formate C-acetyltransferase; D010100:Oxygen; D005998:Glycine