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  The free radical of pyruvate formate-lyase: Characterization by EPR spectroscopy and involvement in catalysis as studied with the substrate-analogue hypopthosphite

Unkrig, V., Neugebauer, F. A., & Knappe, J. (1989). The free radical of pyruvate formate-lyase: Characterization by EPR spectroscopy and involvement in catalysis as studied with the substrate-analogue hypopthosphite. European Journal of Biochemistry, 184(3), 723-728. doi:10.1111/j.1432-1033.1989.tb15072.x.

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Unkrig, Volker, Author
Neugebauer, Franz A.1, Author           
Knappe, Joachim, Author
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1Department of Organic Chemistry, Max Planck Institute for Medical Research, Max Planck Society, ou_1497706              

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 Abstract:

The first-derivative EPR spectrum of the active from 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 formatelyase 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 α-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°C. This finding proves that the enzyme radical is a hydrogen-atom-transferring coenzymic element.

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Language(s): eng - English
 Dates: 1989-06-151989-03-021989-10
 Publication Status: Issued
 Pages: 6
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 Rev. Type: Peer
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Title: European Journal of Biochemistry
Source Genre: Journal
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Publ. Info: Berlin : Published by Springer-Verlag on behalf of the Federation of European Biochemical Societies
Pages: - Volume / Issue: 184 (3) Sequence Number: - Start / End Page: 723 - 728 Identifier: ISSN: 0014-2956
CoNE: https://pure.mpg.de/cone/journals/resource/111097776606040