Subunit stoichiometry and molecular weight of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

Authors:
  • KAK J Angelides
    K J Angelides
  • SAS K Akiyama
    S K Akiyama
  • GHG G Hammes
    G G Hammes
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Abstract
The molar ratio of the component enzymes of the pyruvate dehydrogenase multienzyme complex from E. coli was 1.8:1.7:1 [pyruvate decarboxylase (E1):dihydrolipoyl transacetylase (E2):dihydrolipoyl dehydrogenase (E3)]. This ratio was determined by measuring the Coomassie blue staining of the constituent enzymes after sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. The above ratio is the average of 4 separate experiments with 2 different enzyme preparations. The average MW of the individual enzymes were 96,000, 76,000 and 55,000 for E1, E2 and E3, respectively, by sodium dodecyl sulfate and sodium dodecyl sulfate/8 M urea polyacrylamide gel electrophoresis and by column chromatography in 6 M guanidine .cntdot. HCl. The MW of E2 was reduced to 33,000-36,000 after extensive reduction and alkylation with iodoacetamide. The MW of the complex, E1 and E3 were 4,800,000, 182,000 and 104,000, respectively, using low-angle laser light scattering. Both E1 and E3 are dimeric under the conditions employed. If octahedral symmetry is assumed for the E2 core, a polypeptide chain ratio of 24:24:12 (E1:E2:E3) is in good agreement with the measured molar ratio of component enzymes and the MW of the pyruvate dehydrogenase complex.

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