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Medientyp:
E-Artikel
Titel:
The Free Radical in Pyruvate Formate-Lyase is Located on Glycine-734
Beteiligte:
Frey, Manfred;
Neugebauer, Franz A.;
Schafer, Wolfram;
Knappe, Joachim
Erschienen:
National Academy of Sciences of the United States of America, 1992
Erschienen in:
Proceedings of the National Academy of Sciences of the United States of America, 89 (1992) 3, Seite 996-1000
Sprache:
Englisch
ISSN:
0027-8424
Entstehung:
Anmerkungen:
Beschreibung:
<p>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<sup>13</sup>C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central<sup>13</sup>C nucleus (A<sub/>| = 4.9 mT and A<sub>⊥</sub>= 0.1 mT) and to<sup>13</sup>C nuclei in α and β positions agree with literature data for glycine radical models. N-coupling was verified through uniform<sup>15</sup>N-labeling. The large<sup>1</sup>H hyperfine splitting (1.5 mT) dominating the EPR spectrum was assigned to the α 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.</p>