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Fluorescent derivatives of the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex.

Publication ,  Journal Article
Papadakis, N; Hammes, GG
Published in: Biochemistry
May 3, 1977

One sulfhydryl group per polypeptide chain of the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was selectively labeled with N-[P-(2-benzoxazoyl)phenyl]-maleimide (NBM), 4-dimethylamino-4-magnitude of-maleimidostilbene (NSM), and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) in 0.05 M potassium phosphate (pH 7). Modification of the sulfhydryl group did not alter the enzymatic activity or the binding of 8-anilino-1-naphthalenesulfonate (ANS) or thiochrome diphosphate to the enzyme. The fluorescence of the NBM or NSM coupled to the sulfhydryl group on the enzyme was quenched by binding to the enzyme of the substrate pyruvate the coenzyme thiamine diphosphate, the coenzyme analogue thiochrome diphosphate, the regulatory ligands acetyl-CoA, GTP, and phosphoenolpyruvate, and the acetyl-CoA analogue, ANS. Fluorescence energy transfer measurements were carried out for the enzyme-bound donor-acceptor pairs NBM-ANS, NBM-thiochrome diphosphate ANS-DDPM, and thiochrome diphosphate-DDM. The results indicate that the modified sulfhydryl group is more than 40 A from the active site and approximately 49 A from the acetyl-CoA regulatory site. Thus, a conformational change must accompany the binding of ligands to the regulatory and catalytic sites. Anisotropy depolarization measurements with ANS bound on the isolated pyruvate dehydrogenase in 0.05 M potassium phosphate (pH 7.0) suggest that under these conditions the enzyme is dimeric.

Duke Scholars

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

May 3, 1977

Volume

16

Issue

9

Start / End Page

1890 / 1896

Location

United States

Related Subject Headings

  • Thiazoles
  • Thiamine
  • Pyrimidines
  • Organophosphorus Compounds
  • Maleimides
  • Lipoproteins, VLDL
  • Lipoprotein Lipase
  • Escherichia coli
  • Energy Transfer
  • Biochemistry & Molecular Biology
 

Citation

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Papadakis, N., & Hammes, G. G. (1977). Fluorescent derivatives of the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex. Biochemistry, 16(9), 1890–1896. https://doi.org/10.1021/bi00628a020
Papadakis, N., and G. G. Hammes. “Fluorescent derivatives of the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex.Biochemistry 16, no. 9 (May 3, 1977): 1890–96. https://doi.org/10.1021/bi00628a020.
Papadakis, N., and G. G. Hammes. “Fluorescent derivatives of the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex.Biochemistry, vol. 16, no. 9, May 1977, pp. 1890–96. Pubmed, doi:10.1021/bi00628a020.
Journal cover image

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

May 3, 1977

Volume

16

Issue

9

Start / End Page

1890 / 1896

Location

United States

Related Subject Headings

  • Thiazoles
  • Thiamine
  • Pyrimidines
  • Organophosphorus Compounds
  • Maleimides
  • Lipoproteins, VLDL
  • Lipoprotein Lipase
  • Escherichia coli
  • Energy Transfer
  • Biochemistry & Molecular Biology