The free radical in pyruvate formate-lyase is located on glycine-734
- PMID: 1310545
- PMCID: PMC48372
- DOI: 10.1073/pnas.89.3.996
The free radical in pyruvate formate-lyase is located on glycine-734
Abstract
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.
Similar articles
-
The free radical of the anaerobic ribonucleotide reductase from Escherichia coli is at glycine 681.J Biol Chem. 1996 Mar 22;271(12):6827-31. J Biol Chem. 1996. PMID: 8636106
-
YfiD of Escherichia coli and Y06I of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen-fragmented pyruvate formate-lyase.Biochem Biophys Res Commun. 2001 Jul 13;285(2):456-62. doi: 10.1006/bbrc.2001.5186. Biochem Biophys Res Commun. 2001. PMID: 11444864
-
Electron paramagnetic resonance evidence for a cysteine-based radical in pyruvate formate-lyase inactivated with mercaptopyruvate.Biochemistry. 1995 May 2;34(17):5712-7. doi: 10.1021/bi00017a002. Biochemistry. 1995. PMID: 7727431
-
A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli.FEMS Microbiol Rev. 1990 Aug;6(4):383-98. doi: 10.1111/j.1574-6968.1990.tb04108.x. FEMS Microbiol Rev. 1990. PMID: 2248795 Review.
-
A glycyl radical solution: oxygen-dependent interconversion of pyruvate formate-lyase.Mol Microbiol. 1998 Aug;29(4):945-54. doi: 10.1046/j.1365-2958.1998.00941.x. Mol Microbiol. 1998. PMID: 9767563 Review.
Cited by
-
Pyruvate formate-lyase, evidence for an open conformation favored in the presence of its activating enzyme.J Biol Chem. 2010 Aug 27;285(35):27224-27231. doi: 10.1074/jbc.M109.096875. Epub 2010 Jun 22. J Biol Chem. 2010. PMID: 20571026 Free PMC article.
-
Thermal inactivation of reduced ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli.FEBS Lett. 2002 Oct 9;529(2-3):237-42. doi: 10.1016/s0014-5793(02)03349-5. FEBS Lett. 2002. PMID: 12372607 Free PMC article.
-
Mechanism of Radical S-Adenosyl-l-methionine Adenosylation: Radical Intermediates and the Catalytic Competence of the 5'-Deoxyadenosyl Radical.J Am Chem Soc. 2022 Mar 23;144(11):5087-5098. doi: 10.1021/jacs.1c13706. Epub 2022 Mar 8. J Am Chem Soc. 2022. PMID: 35258967 Free PMC article.
-
Biochemical and physiological characterization of the pyruvate formate-lyase Pfl1 of Chlamydomonas reinhardtii, a typically bacterial enzyme in a eukaryotic alga.Eukaryot Cell. 2008 Mar;7(3):518-26. doi: 10.1128/EC.00368-07. Epub 2008 Feb 1. Eukaryot Cell. 2008. PMID: 18245276 Free PMC article.
-
Biotin synthase from Escherichia coli: isolation of an enzyme-generated intermediate and stoichiometry of S-adenosylmethionine use.Biochem J. 1998 Mar 15;330 ( Pt 3)(Pt 3):1079-85. doi: 10.1042/bj3301079. Biochem J. 1998. PMID: 9494071 Free PMC article.
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases