The Subunit Structure of the Pyruvate‐Dehydrogenase Complex from <i>Escherichia coli</i> K‐12

Vogel, Otto; Hoehn, Barbara; Henning, Ulf

doi:10.1111/j.1432-1033.1972.tb02105.x

Cited by 24 publications

(6 citation statements)

References 22 publications

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“…The results of the re-association assays with separated components, though exhibiting a new lag period, gave no indication for a dissociation of the complex in dilute solution. Sucrose gradient centrifugation of the native complex [25] also speaks against a rapid dissociation after dilution (in fact there is a dissociation of some weaker bound subunits of the pyruvate dehydrogenase component observed during purification, leading to a "core complex" of constant composition [17], but this is meaningless for the experiments done here, because in all cases purified "core complex" was used). Dilution may diminish an intermolecular stabilization of an active enzyme conformation.…”

Section: Discussionmentioning

confidence: 99%

Regulatory Properties of the Pyruvate-Dehydrogenase Complex from Escherichia coli. Thiamine Pyrophosphate as an Effector

Bisswanger

1974

Eur J Biochem

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The pyruvate dehydrogenase complex from Escherichia coli is subject to an allosteric control. Pyruvate shows a homotropic cooperative effect. The extent of the cooperativity increases with decreasing concentrations of thiamine pyrophosphate. This cofactor itself exhibits positive cooperativity in steady-state measurements. The saturation curves both of pyruvate and of thiamine pyrophosphate have a very unusual asymmetric shape. The sigmoidal range is limited to low ligand concentrations and changes to a hyperbolic form far below half saturation. It was demonstrated that allosteric systems with more than eight interacting protomers, which obeys the allosteric model of Monod, J., Wyman, J., and Changeux, J.-P. [J. Mol. Biol. 12 (1965) 88-1171, behave in this manner.In the absence of substrate and thiamine pyrophosphate, the pyruvate dehydrogenase exists in an inactive form. At low thiamine pyrophosphate concentrations addition of pyruvate leads, after a lag phase of several minutes, to a partial activation. Within less than one second, complete activation occurs when saturating amounts of thiamine pyrophosphate are added, irrespective of the presence or absence of pyruvate. The lag period increases with decreasing degree of enzyme dilution. This phenomenon is not due to a dissociation and reassociation of subunits.Oxidative decarboxylation of pyruvate and transfer of the acetyl moiety formed to coenzyme A, corresponding to the overall reaction Pyruvate + NAD' + CoA Dihydrolipoyl-E2 + FAD-E3Reduced FAD-E3 + NAD'where El is pyruvate dehydrogenase, E2 dihydrolipoamide transacetylase, E3 dihydrolipoamide dehydrogenase and TPP, thiamine pyrophosphate. In the complex 16 identical subunits of each enzyme component are arranged to build up the native structure [3].The pyruvate dehydrogenase complex occupies a key position in metabolism as a link between glycolysis and the tricarboxylic acid cycle. Consequently the activity of this enzyme complex is influenced by several glycolytic intermediates [4 -61. Furthermore, the complex is subject to allosteric control, where pyruvate shows a positive cooperative effect, while acetyl-CoA is a strong allosteric inhibitor [7]. The target for nearly all effectors including acetyl-CoA is the pyruvate dehydrogenase component, which exhibits the cooperative effect of pyruvate also in the isolated, presumably Eur. J. Biochem. 48 (1974)

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Section: Discussionmentioning

confidence: 99%

Regulatory Properties of the Pyruvate-Dehydrogenase Complex from Escherichia coli. Thiamine Pyrophosphate as an Effector

Bisswanger

1974

Eur J Biochem

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“…There is a long-lasting discussion on the stoichiometry of the E. coli complex; chain ratios of 2:2:1 [6], 2:l:l [8] and I :I :I [9] have been proposed. It has been recognized that the amounts of El and E, are not constant in the organism, that preparations of the complex show heterogeneity, and that the chain ratio may change during purification due to losses of these components [5,9 -131.…”

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confidence: 99%

The composition of the pyruvate dehydrogenase complex from Azotobacter vinelandii

Bosma¹,

Kok

Westphal

et al. 1984

European Journal of Biochemistry

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An improved purification procedure of the pyruvate dehydrogenase complex of Azotobucter vinelundii is described. This procedure minimizes losses of components and results in the isolation of the pure complex with a specific activity of 15 -19 U/mg and an overall yield of 40 %.The chain ratio of the three components was determined by covalent modification of the lysine residues with trinitrobenzene sulfonic acid, followed by separation of the components on sodium dodecyl sulfate gels. These determinations yielded an average chain ratio of 1.3:3 :0.5 for El :E, :E, respectively. Based on Ez this corresponds with a minimum molecular mass of approximately 216 kDa. Because the molecular mass of the complex has been determined previously to be 800 50 kDa, it is concluded that the complex as isolated from A . vinelundii is based on a tetramer of E2 chains.The complex can be resolved into its individual components, which can be recombined to yield a fully active complex. Titration of E,E, subcomplexes with El resulted in maximum complex activity at an EJE, ratio of 1.5 -1.6. Similar titrations of EIE, subcomplexes with E, resulted in maximum activity at an E,/E, ratio of 0.45 -0.55. From these experiments it is concluded that the complex has maximum activity with a composition of three El dimers, one E, tetramer and one E, dimer. With excess of either El or E, a decrease in activity is observed which indicates competition between these components for binding sites on E,.Asshownbefore a core of 24 E, chains, arranged in a cube with 432 symmetry [l]. Estimates of the molecular mass of this complex vary within 3 -6 MDa [2,4 -71.There is a long-lasting discussion on the stoichiometry of the E. coli complex; chain ratios of 2:2:1 [6], 2:l:l [8] and I :I :I [9] have been proposed. It has been recognized that the amounts of El and E, are not constant in the organism, that preparations of the complex show heterogeneity, and that the chain ratio may change during purification due to losses of these components [5,9 -131. Nevertheless, the E2 core has an inborn capacity for the binding of El and E, dimers. The discrepancy between the reports of several investigators is therefore probably due to a different interpretation of results as obtained by various methods.The pyruvate dehydrogenase complex as isolated from the gram-negative bacterium Azotobucter vinelandii is much smaller than the E. coli complex [14]. Its sedimentation coefficient is 17 -19 S, that of E. coli PDC is 53 -60 S [2,4,7,10]. However, a 17 -20-S form of the E. coli PDC has been observed [7,10] and we have shown that the A . vinelandii complex can be converted into a 56-S form, resembling the E. coli PDC on electron micrographs [15]. 542We have shown recently [I61 that the 56-S form of the A . Ginelandii PDC is an octamer of a 750 -850-kDa particle. The tendency to aggregate is especially pronounced in the isolated E, component; its appearance on electron micrographs is similar to that of E, from E. coli. By sedimentation and light scattering studies it wa5 shown...

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“…In an extension of the studies presented in this thesis, Drs. R. DE ABREU of our laboratory (personal communication) recently estimated that these SDSbands have molecular weights of 90,000; 80,000; 56,000 and 50,000 daltons and in view of the data from the literature (VOGEL et al, 1972b) they probably correspond to PDH, LTA, lipoamide dehydrogenase and PTA respectively. Furthermore, interpretation of the flavin content per mg protein points at a minimal molecular weight of 550,000-620,000 daltons and the latter value agrees with that found for a catalytically-inactive subunit.…”

Section: Pyruvate Dehydrogenase Complex From Azotobacter Vinelandiimentioning

confidence: 94%

“…Like most procedures concerning the isolation and purification of PDC, the one for Azotobacter is also very similar with that developed and improved by REED and WILLMS (1965) and ELEY et al (1972) for E. coli Crookes strain. The group of HENNING is using a different and very elegant procedure, which is not satisfactorily used in our case (VOGEL et al, 1972b). The purification of Azotobacter PDC will be described below in more detail in a four-step scheme and will be referred to as method la.…”

Section: Purification Of the Complex; The 'Pure' Complexmentioning

confidence: 99%

“…Nearest in molecular weight is the enzyme isolated from E. coli K-12, which is, in the hands of DENNERT and HÖGLUND (1970), very labile too. The suggestion made by VOGEL et al (1972b), that this lability could be introduced by the acid fractionation step in the purification procedure, turned out to be by no means valuable in the Azotobacter case, for complexes purified according to method lb are behaving similarly. Active Enzyme Centrifugation might be the method to be applied; the sedimentation coefficient of the enzyme during catalysis can be estimated in crude extracts and after purification (SCHMITT et al, 1971).…”

Section: Meded Landbouwhogeschool Wageningenmentioning

confidence: 99%

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The Pyruvate Dehydrogenase Complex from Azotobacter vinelandii

Kok¹,

Berg²,

Berkel³

et al. 1994

Flavins and Flavoproteins 1993

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The Subunit Structure of the Pyruvate‐Dehydrogenase Complex from Escherichia coli K‐12

Cited by 24 publications

References 22 publications

Regulatory Properties of the Pyruvate-Dehydrogenase Complex from Escherichia coli. Thiamine Pyrophosphate as an Effector

Regulatory Properties of the Pyruvate-Dehydrogenase Complex from Escherichia coli. Thiamine Pyrophosphate as an Effector

The composition of the pyruvate dehydrogenase complex from Azotobacter vinelandii

The Pyruvate Dehydrogenase Complex from Azotobacter vinelandii

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