The stoichiometry of polypeptide chains in the pyruvate dehydrogenase multienzyme complex of <i>E. Coli</i> determined by a simple novel method

Bates, David L.; Harrison, Richard A.; Perham, Richard N.

doi:10.1016/0014-5793(75)80764-2

Cited by 69 publications

(48 citation statements)

References 16 publications

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“…The results obtained from NaDodSO4 and NaDodSO4/urea/polyacrylamide gel electrophoresis and from column chromatography in 6 M guanidine-HCl (both empirical methods) are similar to previously reported molecular weights (1,2) although the values for E1 and E2 are somewhat less than recently reported (4,14 (13). The breakdown of E2 into two approximately equal parts after reduction and alkylation is similar to the results recently reported in which E2 was subjected to limited proteolysis (15,16).…”

Section: Resultssupporting

confidence: 59%

“…The subunit stoichiometry and molecular weight of the complex are consistent with the reported flavin (FAD) content of the complex, 1.8-2.8 nmol/mg (9-13 FAD molecules per complex) (17,18), and with the degree of acetylation (19) and N-ethylmaleimide modification (20)(21)(22) of the lipoic acids, 10 nmol/mg (nt48 acetyl groups and maleimides per complex) (1,18). However, they do not correspond well with the results of Perham and coworkers (14,25) or of Vogel and coworkers (4,26). The reason for these discrepancies is not known.…”

Section: Resultscontrasting

confidence: 54%

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

Angelides¹,

Akiyama²,

Hammes³

1979

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Resultssupporting

confidence: 59%

Section: Resultscontrasting

confidence: 54%

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

Angelides¹,

Akiyama²,

Hammes³

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…Without having determined directly the number of pyruvate dehydrogenase chains and knowing that the variability in the number of these chains, is between 1 and 2 [6,24,25], no choice can be made between the values of two and four lipoyl residues per chain. We arrive therefore either at a chain stoichiometry pyruvate dehydrogenase : lipoyltransacetylase : lipoamide dehydrogenase of 1.…”

Section: Discussionmentioning

confidence: 99%

Determination of the Chain Stoichiometries from the Number of Reactive Sulfhydryl Groups in the Pyruvate Dehydrogenase Complexes of Azotobacter vinelandii and Escherichia coli

Abreu¹,

Kok²,

Graaf-Hess³

et al. 1977

European Journal of Biochemistry

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The pyruvate dehydrogenase complex from Azotobucter vinelundii incubated with 0.05 -0.7 mM [2-14C]pyruvate, magnesium chloride and thiamine pyrophosphate under anaerobic conditions at 0 "C, incorporates four ['4C]acetyl groups per mole FAD which are bound to the highmolecular-weight lipoyl transacetylase. With 10 mM pyruvate, the low-molecular-weight lipoyl transacetylase is also labelled; to this enzyme 3-4 ['4C]acetyl groups are bound per mole FAD. This enzyme is not labelled when pyruvate is used in concentrations lower than 0.7 mM. The complex of A . vinehdii pre-labelled with non-radioactive N-ethylmaleimide did not incorporate label onto low-molecular-weight lipoyl transacetylase; a maximum of four acetyl groups are bound to the high-molecular weight lipoyl transacetylase.Maximum incorporation of four [14C]acetyl groups per mole FAD is found, within five seconds, on the lipoyl transacetylase component when the Escherichiu coli complex is labelled under anaerobic conditions, also in the presence of 10 mM pyruvate.In both complexes all incorporated acetyl groups are bound to sulfhydryl groups since they can be completely removed with hydroxylamine, by performic acid oxidation and by reaction with coenzyme A and arsenite. Under aerobic conditions a slow deacetylation is observed, which is blocked in the presence of N-ethylmaleimide.The multi-enzyme complex from A . vinelundii pre-labelled with non-radioactive N-ethylmaleimide and then labelled with N-ethyl [2,3-1"C]maleimide in the presence of pyruvate, magnesium chloride and thiamine pyrophosphate incorporates under anaerobic conditions at 0 "C, even at 10 mM pyruvate, a maximum of four N-ethyl['4C]maleimide groups per mole of FAD. The label is almost exclusively bound to the high-molecular-weight lipoyl transacetylase of the A . vinrfundii complex.The E. coli complex binds only 2-3 N-ethyl[14C]maleimide groups per mole of FAD under these conditions. Direct labelling and correction for labelling without pyruvate yields 4 -5 N-ethyl-[14C]maleimide per mole FAD.The low-molecular-weight lipoyl transacetylase can be removed by chromatography of the complex from A . vinelundii on a blue-dextran-Sepharose 4B column [De Abreu et ul. (1977) FEBS Lett. 82,89-921. The complex eluted from the column, consisting of the other three enzyme components, binds a maximum of four [14C]acetyl groups per mole of FAD, even with 10 mM pyruvate.It is concluded that the lipoyl/FAD ratio is four in the complexes as isolated by us from both sources. The interpretation of this ratio with respect to the stoichiometries of the different enzyme components will be discussed.The oxidative decarboxylation of pyruvate and the is catalyzed through the following sequence of reactions Pyruvate + thiamine-P2,--C02

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“…Then the E2p transfers the acyl group to CoA while reducing its lipoyl domain, which is then oxidized by the E3 which transfers electrons to NAD + via its cofactor FAD, resulting in the formation of NADH [14]. The formation of PDC not only allows substrate channeling between neighboring enzymes but also connects remote E1p and E3 within the complex by active-site coupling via flexible lipoyl domains of the E2p core [15-17].Attempts to study the PDC has primarily used structure analysis methods such as analytical ultracentrifugation, Cryo-EM, crystallography, isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and neutron scattering [18][19][20][21][22][23][24].Interestingly, structural studies of the PDC have reported varying sizes of the complex [25][26][27][28][29][30].In this study, we used size exclusion chromatography (SEC) to examine the size of PDC in its 2 0…”

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confidence: 99%

The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

Lee

Bhattacharya

et al. 2020

Preprint

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The pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA.PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into submegadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of saltlability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.Keywords: PDC, mitochondrial metabolism, TCA cycle, pyruvate, acetyl-CoA, highperformance liquid chromatography (HPLC), size exclusion chromatography (SEC) synthesis, needs PDC to convert pyruvate into acetyl-CoA [1,2]. Being a key enzyme in cellular metabolism, PDC is also considered a target for anticancer and antibacterial drugs [3,4].Furthermore, the level of nuclear PDC is correlated with that of histone acetylation as well as its recruitment to some promoters upon induction possibly for local acetyl-CoA production [5][6][7][8].The central role of PDC in energy homeostasis necessitates a tight regulation of its activity. Short-term regulation (minutes to hours) occurs through the inhibitory phosphorylation of E1α by pyruvate dehydrogenase kinases (PDKs) and phosphatases, while long-term regulation of PDC activity can be exerted at transcriptional levels within days to weeks [9,10]. PDKs have been studied as potential drug targets as well because of their upregulation or activation in diseases such as diabetes and cancer [11,12]. The catalysis of PDC is performed by three separate enzymes E1p (pyruvate decarboxylase), E2p (dihydrolipoamide acyltransferase), and E3 (dihydrolipoamide dehydrogenase), which are linked together efficiently into a large multienzyme complex, where the oligomeric E2p forms a structural core, to which multiple copies of the E1p, E3, and E3BP (E3 binding protein) are bound [13]. The E1p performs the rate-limiting oxidative decarboxylation of pyruvate via thiamine pyrophosphate (TPP) and transfers the acetyl-group to the lipoyl group of the E2p. Then the E2p transfers the acyl group to CoA while reducing its lipoyl domain, which is then oxidized by the E3 which...

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The stoichiometry of polypeptide chains in the pyruvate dehydrogenase multienzyme complex of E. Coli determined by a simple novel method

Cited by 69 publications

References 16 publications

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

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

Determination of the Chain Stoichiometries from the Number of Reactive Sulfhydryl Groups in the Pyruvate Dehydrogenase Complexes of Azotobacter vinelandii and Escherichia coli

The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

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