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PubMed2003

Prediction of the binding site on E1 in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

The beta-subunit (E1beta) of the pyruvate decarboxylase (E1, alpha(2)beta(2)) component of the Bacillus stearothermophilus pyruvate dehydrogenase complex was comparatively modelled based on the crystal structures of the homologous 2-oxoisovalerate decarboxylase of Pseudomonas putida and Homo sapiens. Based on this homology modelling, alanine-scanning mutagenesis studies revealed that the negatively charged side chain of Glu285 and the hydrophobic side chain of Phe324 are of particular importance in the interaction with the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the complex. These results help to identify the site of interaction on the E1beta subunit and are consistent with thermodynamic evidence of a mixture of electrostatic and hydrophobic interactions being involved.

Alanine↗
PubMed1983

Temperature-dependence of intramolecular coupling of active sites in pyruvate dehydrogenase multienzyme complexes.

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complexes of Escherichia coli, ox heart and Bacillus stearothermophilus was measured at various temperatures. As the temperature was raised, the extent of active-site coupling was found to increase, approaching a maximum near the physiological growth temperature of the organism. Under these conditions, a single pyruvate dehydrogenase (lipoamide) dimer appeared able to cause a rapid (20s) reductive acetylation of probably all 24 polypeptide chains in the dihydrolipoamide acetyltransferase core of the enzyme complex from E. coli at 37 degrees C, and of most if not all of the 60 polypeptide chains in the dihydrolipoamide acetyltransferase cores of the enzymes from ox heart and B. stearothermophilus at 37 degrees C and 60 degrees C respectively. Experiments designed to measure the inter-core and intra-core migration of enzyme subunits suggested that, in the bacterial enzymes at least, this was not a major contributor to active-site coupling.

Acetylation↗
PubMed1988

In utero central nervous system damage in pyruvate dehydrogenase deficiency.

Pyruvate dehydrogenase deficiency is among the most common causes of congenital lactic acidosis. We describe siblings with congenital lactic acidosis due to a deficiency of pyruvate dehydrogenase complex. The findings of computed tomography and pathologic studies suggest that central nervous system damage had occurred in utero. These observations have implications for treatment and outcome in patients with enzymatic defects causing congenital lactic acidosis.

Brain↗
PubMed1982

Growth characteristics of Aspergillus nidulans mutans defective in carbohydrate metabolism.

Several mutants aspergillus nidulans defective in carbohydrate metabolism were tested for growth on different carbon sources. D-Galacturonate was found to be a substrate, useful to discriminate between mutants in pyruvate kinase, pyruvate dehydrogenase complex or pyruvate carboxylase. The results of these tests indicate how particular classes of mutants can be obtained and which substrates can be used preferentially for a rapid phenotypical screening of unknown mutants.

Aspergillus nidulans↗
PubMed1992

Identification and analysis of the genes coding for the putative pyruvate dehydrogenase enzyme complex in Acholeplasma laidlawii.

A monospecific antibody recognizing two membrane proteins in Acholeplasma laidlawii identified a plasmid clone from a genomic library. The nucleotide sequence of the 4.6-kbp insert contained four sequential genes coding for proteins of 39 kDa (E1 alpha, N terminus not cloned), 36 kDa (E1 beta), 57 kDa (E2), and 36 kDa (E3; C terminus not cloned). The N termini of the cloned E2, E1 beta, and native A. laidlawii E2 proteins were verified by amino acid sequencing. Computer-aided searches showed that the translated DNA sequences were homologous to the four subenzymes of the pyruvate dehydrogenase complexes from gram-positive bacteria and humans. The plasmid-encoded 57-kDa (E2) protein was recognized by antibodies against the E2 subenzymes of the pyruvate and oxoglutarate dehydrogenase complexes from Bacillus subtilis. A substantial fraction of the E2 protein as well as part of the pyruvate dehydrogenase enzymatic activity was associated with the cytoplasmic membrane in A. laidlawii. In vivo complementation with three different Escherichia coli pyruvate dehydrogenase-defective mutants showed that the four plasmid-encoded proteins were able to restore pyruvate dehydrogenase enzyme activity in E. coli. Since A. laidlawii lacks oxoglutarate dehydrogenase and most likely branched-chain dehydrogenase enzyme complex activities, these results strongly suggest that the sequenced genes code for the pyruvate dehydrogenase complex.

Acholeplasma laidlawii↗
PubMed1980

Kinetic analysis of the role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The catalytic roles of the two reductively acetylatable lipoic acid residues on each lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli were investigated. Both lipoyl groups are reductively acetylated from pyruvate at the same apparent rate and both can transfer their acetyl groups to CoASH, part-reactions of the overall complex reaction. The complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, conditions that lead to the modification and inactivation of the S-acetyldihydrolipoic acid residues. Modification was found to proceed appreciably faster than the accompanying loss of enzymic activity. The kinetics of the modification were fitted best by supposing that the two lipoyl groups react with the maleimide at different rates, one being modified at approximately 3.5 times the rate of the other. The loss of complex activity took place at a rate approximately equal to that calculated for the modification of the more slowly reacting lipoic acid residue. The simplest interpretation of this result is that only this residue is essential in the overall catalytic mechanism, but an alternative explanation in which one lipoic acid residue can take over the function of another was not ruled out. The kinetics of inactivation could not be reconciled with an obligatory serial interaction between the two lipoic acid residues. Similar experiments with the fluorescent N-[p-(benzimidazol-2-yl)phenyl]maleimide supported these conclusions, although the modification was found to be less specific than with N-ethylmaleimide. The more rapidly modified lipoic acid residue may be involved in the system of intramolecular transacetylation reactions that couple active sites in the lipoate acetyltransferase component.

Escherichia coli↗
PubMed1978

Regulation of brain pyruvate dehydrogenase multienzyme complex.

A number of excellent and comprehensive reviews on various aspects of pyruvate dehydrogenase multienzyme complex have been written recently. The purpose of the present review is to summarize briefly the reaction mechanism and the regulation of this enzyme. Emphasis is put on the most recent literature not covered by previous reviews. Particular attention is also paid to the regulation of brain pyruvate dehydrogenase multienzyme complex, since a number of patients with neuromuscular diseases, such as Friedreich's ataxia, show a decreased rate of pyruvate oxidation.

Animals↗
PubMed1976

Regulation of the pyruvate dehydrogenase activity in the isolated perfused heart of guinea-pigs.

The activity and the interconversion of the between the pyruvate after pyruvate should read: utilization in the perfused hearts and the pyruvate dehydrogenease complex has been measured in the isolated perfused working hearts of guinea-pigs. 1. The pyruvate dehydrogenase complex is transferred into the active form by high work, in anoxia, with 2,4-dinitrophenol and by perfusion without substrate. The rate of interconversion is faster in the perfused heart than in the homogenate. 2. The active form of the pyruvate dehydrogenase complex limits the pyruvate oxidation. There is a close correlation between the pyruvate utilization in the perfused hearts and the pyruvate dehydrogenase of the active form in the homogenates of the same hearts. 3. The "adenylate energy charge" of the cells is considered as the main regulating factor of the interconversion of the pyruvate dehydrogenase complex as seen in experiments with anoxia, dinitrophenol and high work. The inactivation of the pyruvate dehydrogenase complex by acetyl CoA can be overcome by decreasing ATP/ADP ratios.

Acetates↗
PubMed1981

The role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Two lipoic acid residues on each dihydrolipoamide acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were found to undergo oxidoreduction reactions with NAD+ catalysed by the lipoamide dehydrogenase component. It was observed that: (a) 2 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of acetyl-SCoA and NADH; (b) 4 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of NADH; (c) between 1 and 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with acetyl-SCoA plus NADH; (d) 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with pyruvate either before or after many catalytic turnovers through the overall reaction. There was no evidence to support the view that only half of the dihydrolipoic acid residues can be reoxidized by NAD+. However, chemical modification of lipoic acid residues with N-ethylmaleimide was shown to proceed faster than the accompanying loss of enzymic activity under all conditions tested, which indicates that not all the lipoyl groups are essential for activity. The most likely explanation for this result is an enzymic mechanism in which one lipoic acid residue can take over the function of another.

Acetyl Coenzyme A↗
PubMed1993

Three-dimensional structure of the lipoyl domain from Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex.

The structure of the lipoyl domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of nuclear magnetic resonance spectroscopy. A total of 452 nuclear Overhauser effect distance constraints and 76 dihedral angle restraints were employed as the input for the structure calculations, which were performed using a hybrid distance geometry-simulated annealing strategy and the programs DISGEO and X-PLOR. The overall structure of the lipoyl domain (residues 1 to 79 of the dihydrolipoamide acetyltransferase polypeptide chain) is that of a flattened eight-stranded beta-barrel folded around a core of well-defined hydrophobic residues. The lipoylation site, lysine 42, is located in the middle of a beta-turn, and the N and C-terminal residues of the domain are close together in adjacent beta-strands at the opposite end of the molecule. The polypeptide backbone exhibits a 2-fold axis of quasi-symmetry, with the C alpha atoms of residues 15 to 39 and 52 to 76 being almost superimposable on those of residues 52 to 76 and 15 to 39, respectively (root-mean-square deviation = 1.48 A). The amino acid residues at key positions in the structure are conserved among all the reported primary structures of lipoyl domains, suggesting that the domains all fold in a similar way.

Amino Acid Sequence↗
PubMed1979

Inactivation and disassembly of the pyruvate dehydrogenase multienzyme complex from bovine kidney by limited proteolysis with an enzyme from rat liver.

Mammalian pyruvate dehydrogenase multienzyme complex is inactivated when treated with a leupeptin-sensitive enzyme (termed 'inactivase') obtained from rat liver lysosomes. However, the inactivation of the overall reaction does not affect any of the component activities of the enzyme complex. By several methods it is demonstrated that treatment with the inactivase provokes the disassembly of the complex into its constituent enzyme components which, though being enzymatically active when assayed separately, are unable to catalyze the coordinated reaction sequence of pyruvate oxidation. The dissociation occurs as a consequence of limited proteolysis of the lipoate acetyltransferase core of the multienzyme complex. Isolated nicked acetyltransferase retains its complete enzymatic activity and behaves as a high-molecular-weight aggregate. The lipoamide dehydrogenase and pyruvate dehydrogenase components, however, are not cleaved by the inactivase.

Animals↗
PubMed1979

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

The molar ratio of the component enzymes of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was found to be 1.8:1.7:1[pyruvate decarboxylase (E1):dihydrolipoyl transacetylase (E2):dihydrolipoyl dehydrogenase (E3)]. This ratio was determined by measuring the Coomassie blue staining of the constituent enzymes after sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. The above ratio is the average of four separate experiments with two different enzyme preparations. The average molecular weights of the individual enzymes were found to be 96,000, 76,000, and 55,000 for E1, E2, and E3, respectively, by sodium dodecyl sulfate and sodium dodecyl sulfate/8 M urea polyacrylamide gel electrophoresis and by column chromatography in 6 M guanidine . HCl. The molecular weight of E2 was reduced to 33,000-36,000 after extensive reduction and alkylation with iodoacetamide. The molecular weights of the complex, E1, and E3 were found to be 4,800,000, 182,000, and 104,000, respectively, with low-angle laser light scattering. Both E1 and E3 are dimeric under the conditions employed. If octahedral symmetry is assumed for the E2 core, a polypeptide chain ratio of 24:24:12 (E1:E2:E3) is in good agreement with the measured molar ratio of component enzymes and the molecular weight of the pyruvate dehydrogenase complex.

Coloring Agents↗
PubMed1977

Biochemical genetics of the alpha-keto acid dehydrogenase complexes of Escherichia coli K12: isolation and biochemical properties of deletion mutants.

Mutants of Escherichia coli K12 with deletions in the nadC-lpd region of the chromosome were obtained for use in studies on the expression of the ace (pyruvate dehydrogenase complex, specific components) and lpd (lipomide dehydrogenase) genes. These were isolated by selecting spontaneous aroP mutants (lacking the general aromatic amino-acid permease and thus resistant to inhibitory aromatic amino-acid analogues) and screening for auxotrophy due to deletions extending into neighbouring genes. From 2892 isolates tested, the AroP- phenotypes of 2322 were confirmed and, of these, 28 stable and independently-derived auxotrophos were designated as deletion mutants. Six nutritionally-distinct categories were recognized: Nad- (8 strains); Nad-Ace-(7): Nad-'Ace-' (3); Ace- (8); 'Ace-' (I); Lpd-(I). The Ace- phenotypes of four isolates designated 'Ace-' were leaky and enzymological studies confirmed that they had less than 7% of parental pyruvate dehydrogenase complex activity. Enzymological studies showed that the 15 Ace- or Nad-Ace- strains all lacked the pyruvate dehydrogenase complex and pyruvate dehydrogenase (EIp) activities and only three retained detectable dihydrolipoamide acetyltransferase (E2p). The one Lpd- strain lacked pyruvate dehydrogenase, dihydrolipoamide acetyltransferase and lipoamide dehydrogenase (E3) activities as well as the activities of the pyruvate and alpha-ketoglutarate dehydrogenase complexes. The results confirmed the gene order nadC-aroP-aceE-aceF-lpd and indicated that no other essential functions are determined by genes within the nadC-lpd region. Resistance to lactate during growth of pps mutants on acetate was directly related to the specific activity of the pyruvate dehydrogenase complex. None of the deletions promoted the high degree of resistance characteristically associated with constitutive expression of the dehydrogenase complex. Six pps mutants having Ace+ or 'Ace-' phenotypes were more sensitive than the parental strains and expression of their ace operons appeared to be affected; most sensitive were the Ace- strains which lacked pyruvate dehydrogenase complex and phosphoenolpyruvate synthetase activities. The lipoamide dehydrogenase activities of the deletion strains (Lpd+) ranged between 30% and 100% of parental levels indicating that expression of their ace operons appeared to be affected; most sensitive were the Ace- strains which lacked pyruvate dehydrogenase complex and phosphoenolpyruvate synthetase activities. The lipoamide dehydrogenase activities of the deletion strains (Lpd+) ranged between 30% and 100% of parental levels indicating that expression of the lpd gene may be affected by the ace operon but can be independent.

Dihydrolipoamide Dehydrogenase↗
PubMed1987

Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes.

Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented.

Acetoin↗
PubMed1996

Protein-protein interactions in the pyruvate dehydrogenase multienzyme complex: dihydrolipoamide dehydrogenase complexed with the binding domain of dihydrolipoamide acetyltransferase.

BACKGROUND: The ubiquitous pyruvate dehydrogenase multienzyme complex is built around an octahedral or icosahedral core of dihydrolipoamide acetyltransferase (E2) chains, to which multiple copies of pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) bind tightly but non-covalently. E2 is a flexible multidomain protein that mediates interactions with E1 and E3 through a remarkably small binding domain (E2BD). RESULTS: In the Bacillus stearothermophilus complex, the E2 core is an icosahedral assembly of 60 E2 chains. The crystal structure of the E3 dimer (101 kDa) complexed with E2BD (4 kDa) has been solved to 2.6 A resolution. Interactions between E3 and E2BD are dominated by an electrostatic zipper formed by Arg135 and Arg139 in the N-terminal helix of E2BD and Asp344 and Glu431 of one of the monomers of E3. E2BD interacts with both E3 monomers, but the binding site is located close to the twofold axis. Thus, in agreement with earlier biochemical results, it is impossible for two molecules of E2BD to bind simultaneously to one E3 dimer. CONCLUSIONS: Combining this new structure for the E3-E2BD complex with previously determined structures of the E2 catalytic domain and the E2 lipoyl domain creates a model of the E2 core showing how the lipoyl domain can move between the active sites of E2 and E3 in the multienzyme complex.

Acetyltransferases↗
PubMed1995

Three-dimensional structure of a lipoyl domain from the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The structure of a lipoyl domain from the pyruvate dehydrogenase multienzyme complex of Escherichia coli has been determined by means of nuclear magnetic resonance spectroscopy. A total of 549 nuclear Overhauser effect distance restraints, 52 phi torsion angle restraints and 16 slowly exchanging amide protons were employed as input for the structure calculations. These were performed using a combined distance geometry-simulated annealing strategy. The domain is a hybrid between the N and C-terminal halves of the first and third lipoyl domains, respectively, of the dihydrolipoyl acetyltransferase component of the E. coli multienzyme complex, representing residues 1 to 33 and 238 to 289 (wild-type numbering). The lipoyl-lysine residue was also replaced by glutamine. Nonetheless, its structure, two four-stranded beta-sheets forming a flattened beta-barrel, closely resembles that of the lipoyl domain from the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus determined previously. As before, the lipoylation site is physically exposed in a tight turn in one of the beta-sheets, and the N and C-terminal residues are close together at the other end of the molecule in adjacent strands of the other beta-sheet. Another prominently conserved feature of the structure is the 2-fold axis of quasi-symmetry relating the N and C-terminal halves of the domain. Consistent with the high level of sequence similarity between lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes from many different sources, these results confirm that all lipoyl domains are likely to have closely related structures.

Acetyltransferases↗
PubMed1990

Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

A 2641-bp EcoRI fragment of DNA that encodes the C-terminal part of the dihydrolipoyl acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. A 705-bp truncated open reading frame was located at the 5'end of the insert which, together with the 588-bp truncated open reading frame at the 3' end of another EcoRI fragment of B. stearothermophilus DNA previously cloned and sequenced [Hawkins, C. F., Borges, A. & Perham, R. N. (1990) Eur. J. Biochem. 191, 337-446], was identified as the gene, pdhC, encoding the E2 polypeptide chain. Direct sequence analysis of the purified E2 chain confirmed that the two EcoRI fragments are adjoining in the B. stearothermophilus genome. The E3 gene, pdhD, begins just 4 bp downstream from the stop codon of the pdhC gene. The amino acid sequences deduced from the pdhC and pdhD genes correspond to proteins of 427 amino acids (E2, Mr 46,265) and 469 amino acids (E3, Mr 49,193), respectively. Both genes are preceded by potential ribosome-binding sites and the E3 gene is followed by a stemloop structure characteristic of rho-independent transcription terminators. The B. stearothermophilus E2 and E3 chains exhibit substantial sequence similarity with the corresponding subunits of other 2-oxo-acid dehydrogenase multienzyme complexes. The cloning and sequence analysis described here complete the description of the gene cluster (pdhA, B, C and D) which encodes the B. stearothermophilus pyruvate dehydrogenase multienzyme complex.

Acetyltransferases↗
PubMedDate not supplied

[Effect of pyruvate dehydrogenase coenzymes and mitochondrial proteins on the accumulation of [35S]lipoic acid].

Coenzymes introduced in the ratio, peculiar for pyruvate dehydrogenase complex into the medium containing fresh-isolated mitochondria and oxidation substrate--pyruvate increase accumulation of [35S] lipoate by these organelles. This process is highly stimulated by introducing either the only CoA or a coenzyme mixture (CoA, thiamine pyrophosphate, FAD, NAD). Addition of phosphate-extracted components of mitochondria and their protein fraction with coenzymes in the ratio indicated above provides maximum accumulation of [35S] lipoate by liver mitochondria. An equimolar mixture of coenzymes as well as protein components evoke no reliable variations in [35S] lipoate accumulation by albino rat liver mitochondria, while addition of the only thiamine pyrophosphate decreases this accumulation. Reconstruction of multienzyme complexes of coenzymes and apoenzymes on mitochondrion membranes accounts for the results obtained.

Animals↗