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. 1978 Oct;75(10):4877–4880. doi: 10.1073/pnas.75.10.4877

Mechanism of action of the pyruvate dehydrogenase multienzyme complex from Escherichia coli

Kimon J Angelides 1, Gordon G Hammes 1
PMCID: PMC336224  PMID: 368802

Abstract

The extent of cooperativity among the polypeptide chain components in the overall reaction catalyzed by the pyruvate dehydrogenase multienzyme complex from Escherichia coli has been studied. Selective inactivation of the pyruvate dehydrogenase component with thiamin thiazolone pyrophosphate demonstrates that no cooperativity between this component and the overall catalytic reaction occurs: the amount of overall complex activity is directly proportional to the fraction of active pyruvate dehydrogenase component. The transacetylase component has two lipoic acid residues on each of its polypeptide chains that can be modified by N-[3H]ethylmaleimide in the presence of pyruvate and thiamin pyrophosphate. The kinetics of the loss of overall complex activity due to modification of the lipoyl residues on the transacetylase component by maleimide reagents shows that not all lipoic acids are coupled into the overall catalytic reaction and that acyl-group and electron pair transfer involving two or more lipoic acids per catalytic cycle must occur. Finally, full complex activity is found when only half the normal flavin content is present. The results indicate that extensive communication among lipoic acids in acyl-group and electron pair transfer must exist in the normal catalytic mechanism. These results are consistent with the average distances between catalytic sites measured by energy transfer experiments.

Keywords: protein modification, subunit interactions, enzyme mechanisms, flavoprotein

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bates D. L., Danson M. J., Hale G., Hooper E. A., Perham R. N. Self-assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. Nature. 1977 Jul 28;268(5618):313–316. doi: 10.1038/268313a0. [DOI] [PubMed] [Google Scholar]
  2. Bates D. L., Harrison R. A., Perham R. N. The stoichiometry of polypeptide chains in the pyruvate dehydrogenase multienzyme complex of E. coli determined by a simple novel method. FEBS Lett. 1975 Dec 15;60(2):427–430. doi: 10.1016/0014-5793(75)80764-2. [DOI] [PubMed] [Google Scholar]
  3. Collins J. H., Reed L. J. Acyl group and electron pair relay system: a network of interacting lipoyl moieties in the pyruvate and alpha-ketoglutarate dehydrogenase complexes from Escherichia coli. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4223–4227. doi: 10.1073/pnas.74.10.4223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Danson M. J., Perham R. N. Evidence for two lipoic acid residues per lipoate acetyltransferase chain in the pyruvate dehydrogenase multienzyme complex of Escherichia coli. Biochem J. 1976 Dec 1;159(3):677–682. doi: 10.1042/bj1590677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gutowski J. A., Lienhard G. E. Transition state analogs for thiamin pyrophosphate-dependent enzymes. J Biol Chem. 1976 May 10;251(9):2863–2866. [PubMed] [Google Scholar]
  6. KOIKE M., REED L. J. alpha-Keto acid dehydrogenation complexes. II. The role of protein-bound lipoic acid and flavin adenine dinucleotide. J Biol Chem. 1960 Jul;235:1931–1938. [PubMed] [Google Scholar]
  7. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  8. Moe O. A., Jr, Lerner D. A., Hammes G. G. Fluorescence energy transfer between the thiamine diphosphate and flavine adenine dinucleotide binding sites on the pyruvate dehydrogenase multienzyme complex. Biochemistry. 1974 Jun 4;13(12):2552–2557. doi: 10.1021/bi00709a012. [DOI] [PubMed] [Google Scholar]
  9. Reed L. J., Pettit F. H., Eley M. H., Hamilton L., Collins J. H., Oliver R. M. Reconstitution of the Escherichia coli pyruvate dehydrogenase complex. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3068–3072. doi: 10.1073/pnas.72.8.3068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Schwartz E. R., Old L. O., Reed L. J. Regulatory properties of pyruvate dehydrogenase from Escherichia coli. Biochem Biophys Res Commun. 1968 May 10;31(3):495–500. doi: 10.1016/0006-291x(68)90504-4. [DOI] [PubMed] [Google Scholar]
  11. Shepherd G. B., Hammes G. G. Fluorescence energy transfer measurements between ligand binding sites of the pyruvate dehydrogenase multienzyme complex. Biochemistry. 1976 Jan 27;15(2):311–317. doi: 10.1021/bi00647a011. [DOI] [PubMed] [Google Scholar]
  12. Shepherd G. B., Hammes G. G. Fluorescence energy transfer measurements in the pyruvate dehydrogenase multienzyme complex from Escherichia coli with chemically modified lipoic acid. Biochemistry. 1977 Nov 29;16(24):5234–5241. doi: 10.1021/bi00643a012. [DOI] [PubMed] [Google Scholar]
  13. Shepherd G. B., Papadakis N. Fluorescence energy-transfer measurements between coenzyme A and flavin adenine dinucleotide binding sites of the Escherichia coli pyruvate dehydrogenase multienzyme complex. Biochemistry. 1976 Jun 29;15(13):2888–2893. doi: 10.1021/bi00658a029. [DOI] [PubMed] [Google Scholar]
  14. Speckhard D. C., Ikeda B. H., Wong S. S., Frey P. A. Acetylation stoichiometry of Escherichia coli pyruvate dehydrogenase complex. Biochem Biophys Res Commun. 1977 Jul 25;77(2):708–713. doi: 10.1016/s0006-291x(77)80036-3. [DOI] [PubMed] [Google Scholar]

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