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 Escherichia coli was found to be 1 and 2). The enzyme stoichiometry in the complex is controversial: both 2:2:1 (1) and 1-2:1:1 (3, 4) (E1:E2:E3) have been proposed. The E2 forms a structural core to which El and E3 are bound, and electron microscopy and x-ray diffraction measurements suggest that the E2 core has octahedral symmetry (1, 2, 5). The stoichiometry is a fundamental characteristic of the complex that must be known before an understanding of the structure and mechanism can be obtained in molecular terms.In this work, we have prepared high purity E1, E2, and E3. The molecular weights of these enzymes have been determined by sodium dodecyl sulfate (NaDodSO4) and NaDodSO4/ urea/polyacrylamide gel electrophoresis and by column chromatography in guanidine-HCl. The molecular weights of the complex, El, and E3 have also been determined with lowangle laser light scattering. The molar ratio of enzymes in the complex has been obtained by use of NaDodSO4/polyacrylamide slab gel electrophoresis and Coomassie blue staining. The results are consistent with a polypeptide chain ratio of 24:24:12 (E1:E2:E3) and a molecular weight of 4.8 X 106 for the intact complex.MATERIALS AND METHODS Chemicals. The sources of the chemicals were as follows: dithiothreitol, 2-mercaptoethanol, ethanolamine, Sepharose 6B, and hydroxylapatite from Sigma; NaDodSO4, Coomassie blue R-250, Bio-Gel A-5m and A-1.5m from Bio-Rad; ultrapure guanidine-HCI, urea, and ammonium sulfate (special enzyme grade) from Schwarz/Mann; acrylamide, N,N'-methylene bisacrylamide, N,N,N',N'-tetraethylenediamine, iodoacetic acid, and iodoacetamide from Eastman Kodak; NCS tissue solubilizer from Amersham. The phosphorylase a (rabbit muscle), bovine serum albumin, rabbit gamma globulin, transferrin, and cytochrome c were from Sigma; E. coli RNA polymerase (core enzyme), E. coli (K-12) 3-galactosidase, rabbit muscle pyruvate kinase, yeast alcohol dehydrogenase, and soybean trypsin inhibitor were from Boehringer Mannheim. All other chemicals were the best available commercial grades. Solutions were prepared with deionized distilled water.Pyruvate Dehydrogenase Complex. The pyruvate dehydrogenase complex was prepared by the method of Reed and Willms (6). The specific acitivity as measured by the NAD+ reduction assay was between 36 and 42 ,umol of NADH/min per mg of protein at 300 C.Preparation of Subunits. All operations were carried out at 4°C unless otherwise specified, and 3 mM sodium azide was added to all buffers to control microbial growth in prolonged column runs.A solution of pyruvate dehydrogenase complex, approximately 25 mg/ml, was dialyzed against 20 mM ethanolamine/20 mM potassium phosphate (pH 10.0) for 2 hr. The enzyme complex was applied to a Sepharose 6B column (2.5 cm inside diameter X 90 cm) preequilibrated with 20 mM ethanolamine/phosphate (pH 9.5). The protein was eluted with the same buffer, and the eluate was monitored by measurement of the a...

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