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

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.