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PubMed1979

Subunit structure of dihydrolipoyl transacetylase component of pyruvate dehydrogenase complex from Escherichia coli.

Limited tryptic digestion of the pyruvate dehydrogenase complex of Escherichia coli or its dihydrolipoyl transacetylase core cleaves the trypsin-sensitive transacetylase subunits into two large fragments, A (lipoyl domain) and D (subunit binding domain). Release of fragments A from the complex does not significantly affect its sedimentation coefficient or its appearance in the electron microscope. Fragment A contains the lipoyl moieties ((3)H-labeled), is acidic with an apparent isoelectric point of about 4.0, has a M(r) of 31,600 as determined by sedimentation equilibrium analysis, and has a swollen or extended structure (f/f(o) = 1.78). Fragment A exhibits anomalous properties, probably due to its acidic nature. It is resistant to staining with Coomassie blue and it migrates on sodium dodecyl sulfate/polyacrylamide gels as if it had a M(r) of 46,000-48,000. Further tryptic digestion converts fragment A into a lipoyl-containing fragment of M(r) 20,000 (fragment B) and eventually into an apparently stable product of estimated M(r) about 10,000 (fragment C). Fragment D has a compact structure of M(r) about 29,600 as determined by sedimentation equilibrium analysis in 6 M guanidinium chloride, and it possesses the intersubunit binding sites of the transacetylase, the binding sites for pyruvate dehydrogenase and dihydrolipoyl dehydrogenase, and the catalytic site for transacetylation. The assemblage of fragments D is responsible for the cube-like appearance of the transacetylase in the electron microscope. High-resolution electron micrographs of the transacetylase show fiber-like extensions, apparently corresponding to tryptic fragment A, surrounding the cube-like core.

Acetyltransferases↗
PubMed1978

Demonstration of a lag period in the time-course of the reaction catalyzed by pyruvate dehydrogenase complex.

At low thiamine pyrophosphate concentrations the time-course of the reaction catalyzed by mammalian pyruvate dehydrogenase complex shows a lag period of some minutes when the reaction is started by either enzyme, pyruvate or thiamine pyrophosphate. However, started by CoASH or NAD+, the lag period disappears. An increase in enzyme concentration to 25 mU/ml causes a concomitant shortening of the duration of the lag period (tau), while above this value tau is independent of the enzyme concentration. An increase in thiamine pyrophosphate concentration decreases the value of tau and the lag period vanishes at infinite thiamine pyrophosphate concentration. It is suggested that both isomerization and aggregation-dissociation reactions may play an improtant role in the development of the lag period of pyruvate dehydrogenase complex.

Animals↗
PubMed1978

Affinity chromatography studies with the pyruvate dehydrogenase complex of wild-type Escherichia coli.

1. The lifetime of thiamine pyrophosphate-Sepharose 2B affinity matrices synthesized according to Matsuura et al. (Matsuura, A., Iwashina, A. and Nose, Y. (1973) Biochem. Biophys. Res. Commun. 51, 241-246) has been improved. The matrix interacts with bacterial pyruvate dehydrogenase complexes. 2. The synthesis of a stable thiochrome-Sepharose 2B matrix is described. 3. Both matrices bind the pyruvate dehydrogenase complex of Escherichia coli in a 50 mM phosphate buffer, pH 7.0. Elution is possibly by an increase in ionic strength but not by the cofactor or metal-cofactor complexes. 4. The presence of Mg2+, reduces the capacity of the affinity matrices but leads to higher specificity for the multienzyme complex. 5. The pyruvate dehydrogenase complex of E. coli has been successfully purified by combining a classical purification step with these affinity chromatography systems. The method is less suitable for large scale operation.

Chromatography, Affinity↗
PubMed1975

Studies on the physiological significance of the lack of a pyruvate dehydrogenase complex in Hyphomicrobium sp.

Hyphomicrobium X was grown in media containing either methanol or ethanol as a carbon and energy source, with or without additional organic carbon sources. The organism transported pyruvate, malate and succinate into the cells, and incorporated their carbon skeletons into cellular material, but when each of these compounds was added as sole carbon and energy source none supported growth of the organism. Enzymic analysis of crude cell-free extracts failed to detect either a complete pyruvate dehydrogenase complex or an active E1 component. Furthermore, oxygen uptake experiments with whole cell suspensions did not show any oxidation of pyruvate, succinate or malate. The distribution of radioactivity amongst the amino acids in hydrolysates of cell protein obtained from organisms grown in the presence of [14C]pyruvate, [14C]acetate or [14C]succinate indicated that the organism is limited in its ability to metabolize pyruvate. Growth in the presence of [14C]pyruvate resulted in 93% of the total radioactivity recovered being associated with amino acids derived directly from pyruvate. In contrast, growth in the presence of [14C]acetate or [14C]succinate resulted in more-or-less uniform labelling of all biogenic classes of amino acids. These results are consistent with the lack of an active pyruvate dehydrogenase complex which would make it impossible for Hyphomicrobium X to convert pyruvate into acetyl-CoA and to generate energy from carbon compounds for which the energy metabolism relies on oxidation through tricarboxylic acid (TCA) cycle intermediates.

Acetates↗
PubMed1978

Rapid intramolecular coupling of active sites in the pyruvate dehydrogenase complex of Escherichia coli: mechanism for rate enhancement in a multimeric structure.

In the absence of CoA and presence of pyruvate, the lipoic acid residues covalently bound to the lipoate acetyltransferase core component (acetyl-CoA:dihydrolipoate S-acetyltransferase, EC 2.3.1.12) of the pyruvate dehydrogenase multienzyme complex of Escherichia coli become reductively acetylated. A study of a series of reassembled complexes varying only in their content of pyruvate decarboxylase [pyruvate:lipoate-oxidoreductase (decarboxylating and acceptor-acetylating) EC 1.2.4.1] showed that the initial direct reductive acetylation of lipoic acid residues can be followed by extensive intramolecular transacetylation reaction between lipoic acid residues on neighboring polypeptide chains of the lipoate acetyltransferase core [Bates, D. L., Danson, M. J., Hale, G., Hooper, E. A. & Perham, R. N. (1977) Nature (London) 268, 313-316]. Pulsed-quenched-flow measurements of the rates of the acetylation reactions in the various complexes now demonstrate that the intramolecular transacetylation reactions are not rate-determining in the normal reaction mechanism of the enzyme. There is therefore the potential for rapid multiple coupling of active sites in the lipoate acetyltransferase core. The rate constant for the overall complex reaction, measured by stopped-flow fluorimetry, is found to be approximately twice that for the reductive acetylation reaction measured by pulsed-quenched flow. This result could mean that CoA is an allosteric stimulator of the reductive acetylation part of the overall reaction or that there are two active sites on each chain of the lipoate acetyltransferase component working in parallel. A system of rapid functional connection of active sites in a multienzyme complex ensures that sequential reactions can be successfully coupled even under conditions of low substrate concentrations for the different steps. The substantial rate enhancement thus achieved offers a plausible explanation for the unusual complexity of the quaternary structure of the enzyme.

Binding Sites↗
PubMed1979

Defective activation of the pyruvate dehydrogenase complex in subacute necrotizing encephalomyelopathy (Leigh disease).

Autopsy examination confirmed the diagnosis of subacute necrotizing encephalomyelopathy (SNE) in a 7-month-old male infant who underwent several metabolic studies before death. Intermittent lactic acidemia and fumaric aciduria, an extreme hyperglycemic response to an intravenous bolus of alanine, and an elevated total body flux rate of glucose (58.4 mumoles . kg-1 . min-1) suggested a disturbance in the oxidative decarboxylation of pyruvate. Enzymological studies of postmortem samples revealed low nonactivated pyruvate dehydrogenase activity in liver (19.4%) and brain (53.8%). The lowest brain pyruvate dehydrogenase activities were noted in the midbrain and pontine regions. Supramaximal activation of the hepatic pyruvate dehydrogenase complex (135% of control values) occurred in vitro. Spontaneous reactivation following in vitro inactivation of the complex with adenosine triphosphate was significantly less (p less than 0.02) in the patient's samples compared to controls. The biochemical defect was not apparent in fibroblasts. These enzymological observations point to an in vivo defect in the activation mechanism of the pyruvate dehydrogenase complex as the biochemical disturbance in SNE. The findings suggest that dichloroacetate may be beneficial in treating SNE.

Alanine↗
PubMed1977

Fluorescent derivatives of the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex.

One sulfhydryl group per polypeptide chain of the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was selectively labeled with N-[P-(2-benzoxazoyl)phenyl]-maleimide (NBM), 4-dimethylamino-4-magnitude of-maleimidostilbene (NSM), and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) in 0.05 M potassium phosphate (pH 7). Modification of the sulfhydryl group did not alter the enzymatic activity or the binding of 8-anilino-1-naphthalenesulfonate (ANS) or thiochrome diphosphate to the enzyme. The fluorescence of the NBM or NSM coupled to the sulfhydryl group on the enzyme was quenched by binding to the enzyme of the substrate pyruvate the coenzyme thiamine diphosphate, the coenzyme analogue thiochrome diphosphate, the regulatory ligands acetyl-CoA, GTP, and phosphoenolpyruvate, and the acetyl-CoA analogue, ANS. Fluorescence energy transfer measurements were carried out for the enzyme-bound donor-acceptor pairs NBM-ANS, NBM-thiochrome diphosphate ANS-DDPM, and thiochrome diphosphate-DDM. The results indicate that the modified sulfhydryl group is more than 40 A from the active site and approximately 49 A from the acetyl-CoA regulatory site. Thus, a conformational change must accompany the binding of ligands to the regulatory and catalytic sites. Anisotropy depolarization measurements with ANS bound on the isolated pyruvate dehydrogenase in 0.05 M potassium phosphate (pH 7.0) suggest that under these conditions the enzyme is dimeric.

Anilino Naphthalenesulfonates↗
PubMed1976

Symmetry and asymmetry of the pyruvate dehydrogenase complexes from Azotobacter vinelandii and Escherichia coli as reflected by fluorescence and spin-label studies.

Fluorescence-lifetime measurements of FAD bound to lipoamide dehydrogenase from Azotobacter vinelandii and Escherichia 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. vinelandii. Reduction of the complex with Mg2+, 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 [N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide] introduced in such a way proves the existence of at least two different micro-environments around the lipoyl moieties in complex isolated from A. vinelandii. Electron paramagnetic resonance spectra of the specifically spin-labelled complexes from E. coli and A. vinelandii, when dissolved in tricine [N-tris(hydroxymethyl)-methylglycine] buffer, show interactions of at least two electron spins with each other, which indicate that the lipoyl moieties are rather close together. Fluorescent label [N-(1-anilinonaphthyl-4)maleimide] is specifically attached to the lipoyl moiety of the high-Mr transacetylase of the freshly isolated complex from A. vinelandii. From the large differences in the apparent lifetimes tau p and tau m, as detected by phase fluorimetry, it is shown that this fluorscent label is distributed in different micro-environments. The differences observed in energy transfer between fluorescent label, attached to the lipoyl moiety of the high-Mr 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. vinelandii are both non-symmetrically distributed within the complex.

Acetyltransferases↗
PubMed1978

Fluorescence energy-transfer studies on the pyruvate dehydrogenase complex isolated from Azotobacter vinelandii.

Fluorescence energy transfer has been employed to estimate the minimum distance between each of the active sites of the 4 component enzymes of the pyruvate dehydrogenase multienzyme complex from Azotobacter vinelandii. No energy transfer was seen between thiochrome diphosphate, bound to the pyruvate decarboxylase active site, and the FAD of the lipoamide dehydrogenase active site. Likewise, several fluorescent sulfhydryl labels, which were specifically bound to the lipoyl moiety of lipoyl transacetylase, showed no energy transfer to either the flavin or thiochrome diphosphate. These observations suggest that all the active centers of the complex are quite far apart (greater than or equal to 40 nm), at least during some stages of catalysis. These results do not preclude the possibility that the distances change during catalysis. Several of the fluorescent probes used possessed multiple fluorescent lifetimes, as shown by determination of lifetime averages by both phase and modulation measurements on a phase fluorimeter. These lifetimes are shown to result from multiple factors, not necessarily related to multiple protein conformations.

Acetylcysteine↗
PubMed1976

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.

1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...

Acetaldehyde↗