The Pyruvate‐Dehydrogenase Complex from <i>Azotobacter vinelandii</i>

Hj, Grande; Tw, Bresters; Abreu, Ronney A. De; A, de Kok; Veeger, C.

doi:10.1111/j.1432-1033.1975.tb02462.x

Cited by 24 publications

(25 citation statements)

References 20 publications

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“…Assuming four acetyl binding sites on both transacetylase chains, 3.9 -4.4 pyruvate dehydrogenase chains can be accomodated in the four-component complex. This value seems to be in good agreement with the number of Mn2+ binding sites reported earlier [19]. However, it is difficult to estimate accurately the actual number of binding sites from a non-linear Scatchard plot which shows at least three types of MnZ+ binding sites.…”

Section: Discussionsupporting

confidence: 78%

“…The specificity of N-ethylmaleimide analogues for the lipoyl transacetylase component has been demonstrated by Grande et al [19,20] and Brown and Perham [21] and is confirmed in this report. The demonstration that nearly a 1 :1 ratio is obtained by using both labelled substrate and SH reagent confirms the '[able 5.…”

Section: Discussionsupporting

confidence: 76%

“…The incorporation of four N-ethylmaleimide groups per mole of FAD into the three-component complex from A . vineluandii is consistent with the incorporation of 3 -4 spin labels per mole of FAD we reported earlier from experiments with spin label N-ethylmaleimide, derivatives which are like fluorescence labels presumably bound to the high-molecular-weight transacetylase [19]. Here again either two or four lipoyl residues could be present per chain.…”

Section: Discussionsupporting

confidence: 71%

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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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Section: Discussionsupporting

confidence: 78%

Section: Discussionsupporting

confidence: 76%

Section: Discussionsupporting

confidence: 71%

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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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“…Prior to labelling the complex was treated with 1 mM N-ethylmaleimide as described previously [4]. Sodium dodecylsulphate gels without staining were used to determine the position of covalently bound fluorescent label.…”

Section: Methodsmentioning

confidence: 99%

“…there are two different transacetylase components in the A . vinelundii complex [4] and there is one such a component in the E. coli complex [3] as well as the much lower molecular weight and FAD content of the A . vinelundii complex.…”

mentioning

confidence: 99%

Symmetry and Asymmetry of the Pyruvate Dehydrogenase Complexes from Azotobacter vinelandii and Escherichia coli as Reflected by Fluorescence and Spin‐Label Studies

Grande¹,

Telgen²,

Veeger³

1976

European Journal of Biochemistry

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Fluorescence-lifetime measurements of FAD bound to lipoamide dehydrogenase from Azotohucter vineluizdii and Escherichiu coli were performed. It is shown from these results that the two FAD groups in the isolated dimeric enzyme, as well as in the enzyme in the intact complex of E. coli, are in non-equivalent surroundings. This contrasts with the near equivalence of the FAD groups of both the enzyme and complex isolated from A . vinelundii. Reduction of the complex with Mg", thiamine pyrophosphate and pyruvate or with NADH enables the attachment of a maleimide analogue specifically to the lipoyl moieties of the transacetylase(s). Spin label as detected by phase fluorimetry, it is shown that this fluorescent label is distributed in different micro-environments. The differences observed in energy transfer between fluorescent label, attached to the lipoyl moiety of the high-M, transacetylase, indicate different conformations of the complex from A . vinelandii. Upon introduction of the label after reduction with NADH a much larger energy transfer, thus a shorter distance, is observed between the label and FAD than when reduction is performed with Mg2+, thiamine pyrophosphate and pyruvate. A similar conformation dependence upon reduction is found for the pyruvate dehydrogenase complex from E. coli. It is thus proposed that the transacetylase of E. coli and the high-Mr transacetylase of A . vineluizdii are both non-symmetrically distributed within the complex.Earlier studies from this laboratory on the flavoprotein lipoamide dehydrogenase from pig heart [I ] showed that this dimeric enzyme containing identical peptide chains has different fluorescence lifetimes of its two FAD molecules. This indicates that the two prosthetic groups are in non-equivalent surroundings. The lower symmetry can arise from mutual interaction of the FAD molecules and is then of local nature. It is also possible that the symmetry of the enzyme peptide chains is affected by dimerization. This could be induced by the separation of lipoamide dehydrogenase from the complex. Therefore studies on the intact complex were performed. For this purpose the fluorescence lifetimes of the pyruvate dehydrogenase complexes from pig heart and Azotobucter vinelundii were measured at two temperatures. It appeared [2] that the lipoamide dehydrogenase from pig heart in the intact complex has also two non-equivalent FAD groups. This proves that the observed asymmetry is not induced during the purification procedure. The observation that the complex from A . vinelundii did not show this asymmetry of the FAD groups [2] indicated that the non-equivalence is perhaps typical for eucaryotic species.In the present studies properties of the lipoamide dehydrogenase in the pyruvate dehydrogenase complexes from A . vinelundii and Escherichiu coli were

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Regulatory properties of the pyruvate dehydrogenase complex of Pseudomonas aeruginosa

Ghosh

Guest

Jeyaseelan

1981

Biochimica et Biophysica Acta (BBA) - Enzymology

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The Pyruvate‐Dehydrogenase Complex from Azotobacter vinelandii

Cited by 24 publications

References 20 publications

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

Symmetry and Asymmetry of the Pyruvate Dehydrogenase Complexes from Azotobacter vinelandii and Escherichia coli as Reflected by Fluorescence and Spin‐Label Studies

Regulatory properties of the pyruvate dehydrogenase complex of Pseudomonas aeruginosa

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