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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↗
PubMed1999

The inhibitory effects of lipoic compounds on mammalian pyruvate dehydrogenase complex and its catalytic components.

To examine the stereospecific effects of lipoic compounds on pyruvate metabolism, the effects of R-lipoic acid (R-LA), S-lipoic acid (S-LA) and 1,2-diselenolane-3-pentanoic acid (Se-LA) on the activities of the mammalian pyruvate dehydrogenase complex (PDC) and its catalytic components were investigated. Both S-LA and R-LA markedly inhibited PDC activity; whereas Se-LA displayed inhibition only at higher concentrations. Examination of the effects on the individual catalytic components indicated that Se-LA inhibited the pyruvate dehydrogenase component; whereas R-LA and S-LA inhibited the dihydrolipoamide acetyltransferase component. The three lipoic compounds lowered dihydrolipoamide dehydrogrenase (E3) activity in the forward reaction by about 30 to 45%. The kinetic data of E3 showed that both R-LA and Se-LA are used as substrates by E3 for the reverse reaction. Decarboxylation of [1-14C]pyruvate via PDC by cultured HepG2 cells was not affected by R-LA, but moderately decreased with S-LA and Se-LA. These findings indicate that (i) purified PDC and its catalytic components are affected by lipoic compounds based on their stereoselectivity; and (ii) the oxidation of pyruvate by intact HepG2 cells is not inhibited by R-LA. The later finding with the intact cells is in support of therapeutic role of R-LA as an antioxidant.

Acetyltransferases↗
PubMed1994

Pyruvate dehydrogenase complexes from the equine nematode, Parascaris equorum, and the canine cestode, Dipylidium caninum, helminths exhibiting anaerobic mitochondrial metabolism.

The pyruvate dehydrogenase complex (PDC) has been purified to apparent homogeneity from 2 parasitic helminths exhibiting anaerobic mitochondrial metabolism, the equine nematode, Parascaris equorum, and the canine cestode, Dipylidium caninum. The P. equorum PDC yielded 7 major bands when separated by SDS-PAGE. The bands of 72, 55-53.5, 41 and 36 kDa corresponded to E2, E3, E1 alpha and E1 beta, respectively. The complex also contained additional unidentified proteins of 43 and 45 kDa. Incubation of the complex with [2-14C]pyruvate resulted in the acetylation of only E2. These results suggest that the P. equorum PDC lacks protein X and exhibits an altered subunit composition, as has been described previously for the PDC of the related nematode, Ascaris suum. In contrast, the D. caninum PDC yielded only four major bands after SDS-PAGE of 59, 58, 39 and 34 kDa, which corresponded to E3, E2, E1 alpha and E1 beta, respectively. Incubation of the D. caninum complex with [2-14C]pyruvate resulted in the acetylation of E2 and a second protein which comigrated with E3, suggesting that the D. caninum complex contained protein X and had a subunit composition similar to PDCs from other eukaryotic organisms. Both helminth complexes appeared less sensitive to inhibition by elevated NADH/NAD+ ratios than complexes isolated from aerobic organisms, as would be predicted for PDCs from organisms exploiting microaerobic habitats. These results suggest that although these helminths have similar anaerobic mitochondrial pathways, they contain significantly different PDCs.

Acetylation↗
PubMed1996

Sepsis-induced alterations in pyruvate dehydrogenase complex activity in rat skeletal muscle: effects on plasma lactate.

The pyruvate dehydrogenase (PDH) complex undergoes reversible phosphorylation catalyzed by a PDH kinase (inactivating) and a PDH phosphatase (activating). In skeletal muscle, a decreased proportion of PDH complex in the active, nonphosphorylated form (PDHa) limits glucose oxidation and promotes the conversion of pyruvate to lactate. Increased lactate formation with the accompanying hyperlactatemia is a frequent metabolic complication of sepsis. The time course for inactivation of the PDH complex in skeletal muscle during sepsis was contrasted with changes in PDHa during sterile inflammation 3,7, or 14 days following the implantation of the foreign body nidus. Total PDH complex activity was not altered in any of the conditions examined. Sepsis, but not sterile inflammation, caused a reduction in the muscle PDHa measured 3 or 7 days following induction of sepsis. The inhibition of the muscle PDHa during sepsis was associated with a sustained hyperlactatemia. PDH kinase activity measured in extracts of mitochondria was enhanced twofold during this period. Fourteen days after induction of sepsis, there were no differences in the PDHa or plasma lactate concentrations in septic rats compared with either control or sterile inflammation. Furthermore, the PDH kinase activity was decreased to values observed in control values. The results are consistent with the hypothesis that a reduced PDHa in skeletal muscle during sepsis is responsible, in part, for the hyperlactatemia characteristic of septic hypermetabolism. Furthermore, the results provide evidence that the decrease in PDHa results from a stable stimulation of PDH kinase activity.

Abdominal Abscess↗
PubMed1990

Time-resolved fluorescence studies on mutants of the dihydrolipoyl transacetylase (E2) component of the pyruvate dehydrogenase complex from Azotobacter vinelandii.

Fluorescence anisotropy decays were measured for the wild-type dihydrolipoyl transacetylase (E2) component of pyruvate dehydrogenase complex from Azotobacter vinelandii and E. coli and for E2-mutants from A. vinelandii in which the alanine-proline-rich sequence between the binding domain and the catalytic domain is partially or completely deleted. In both E2-mutants the rotational mobility of the lipoyl domain and the overall activity after reconstitution of the complex are significantly decreased indicating the important role of the deleted sequence for the movement of the lipoyl domain and the transfer of substrates between the different active sites within the complex.

Acyltransferases↗
PubMed1989

Structural organization of the gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex.

The structural organization of the X-linked gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex has been determined by restriction endonuclease mapping and DNA sequence analysis of overlapping genomic clones. The gene is approximately 17 kilobase pairs long. It contains 11 exons ranging from 61 to 174 base pairs and introns ranging from 600 base pairs to 5.7 kilobase pairs. All the splice donor and acceptor sites conform to the GT/AG rule. The transcription initiation site was determined by S1 nuclease mapping. The DNA sequence around this site is very GC-rich. A "TATA box"-like sequence and a "CAAT box"-like sequence are present 24 and 113 bases upstream from the cap site, respectively. Also upstream from the cap site are several sets of inverted repeats, direct repeats, several sequences resembling the transcription factor Sp1 binding site, a glucocorticoid-responsive element, and two cAMP receptor binding sites.

Amino Acid Sequence↗
PubMed2004

Immunochemical identification of coenzyme Q0-dihydrolipoamide adducts in the E2 components of the alpha-ketoglutarate and pyruvate dehydrogenase complexes partially explains the cellular toxicity of coenzyme Q0.

Coenzyme Q(0) (Q(0)), a strong electrophile, is toxic to insulin-producing cells. Q(0) was incubated with rat and human pancreatic islets and INS-1 insulinoma cells, and its attachment to cellular proteins was studied with Western analysis using antiserum raised against the benzoquinone ring structure of ubiquinone (anti-Q). Q(0) covalently bonded to two proteins, one of 50 kDa and another of 70 kDa. Both proteins were found to be mitochondrial in human and rat islet cells and in many rat organs. Mitochondria were incubated with Q(0), and affinity-purified anti-Q was used to immunoprecipitate the 50-kDa protein. Amino acid sequencing identified it as dihydrolipoamide succinyltransferase, the E2 component of the alpha-ketoglutarate dehydrogenase complex (KDC). Western analysis also showed that Q bonds to the E2 components of the purified KDC and (0)the pyruvate dehydrogenase complex (PDC). Dihydrolipoamide acetyltransferase, the E2 of the PDC, has a molecular mass of 70 kDa, and the 70-kDa protein was inferred to be this enzyme. Q(0) was found to bond only to proteins containing dihydrolipoate, and in preparations of mitochondria, thiol reducing agents facilitated the attachment of Q(0), but oxidizing agents prevented it, suggesting that Q(0) bonds to thiols of dihydrolipoamide. Incubation of human or pig PDC with Q(0) followed by matrix-assisted laser desorption ionization time-of-flight and liquid chromatography/electrospray ionization mass spectrometry analyses of chymotrypsin-digested peptides of PDC E2 confirmed that Q(0) bonds to the dihydrolipoamide in these proteins. In mitochondria, coenzymes Q(1) and Q(2) did not bond to the 50-kDa protein but competed with the bonding of Q(0) to this protein. The prevention by Q(1) of characteristics the bonding of Q(0) to KDC E2, as well as other of the Q(0) effect, are reminiscent of the action of Q(0) on the mitochondrial permeability transition pore described previously (Fontaine, E., Ichas, F., and Bernardi, P. (1998) J. Biol. Chem. 273, 25734-25740).

Acetyltransferases↗
PubMed1983

Hybrid plasmids containing the pyruvate dehydrogenase complex genes and gene-DNA relationships in the 2 to 3 minute region of the Escherichia coli chromosome.

A sample of colonies from the Clarke-Carbon ColE1-Escherichia coli DNA plasmid gene bank was screened by conjugation for complementation of the lipoamide dehydrogenase lesion of a deletion strain lacking all components of the pyruvate dehydrogenase complex, delta (aroP aceE aceF lpd). Two ColE1-lpd+ hybrid plasmids were identified: pGS2 (ColE1-ace lpd+; 24 kb) and pGS5 (ColE1-lpd+; 14 kb). Enzymological studies confirmed that pGS2 expressed all the activities of the pyruvate dehydrogenase complex, whereas pGS5 expressed the lipoamide dehydrogenase and acetyltransferase activities (the latter from a ColE1 promoter). These and other plasmids were used to construct a 47-site (15 enzymes) restriction map for a 24.2 kb segment of bacterial DNA in the nadC-lpd region. A further 13 sites (six enzymes) were defined in a 5.4 kb sub-segment containing the lpd gene. lambda phage derivatives containing specific fragments were constructed and used in transduction studies which located the ace and lpd genes in a 7.78 kb sub-segment flanked by AccI and NruI sites.

Bacteriophage lambda↗
PubMed1996

In vivo incorporation of lipoic acid enantiomers and homologues in the pyruvate dehydrogenase complex from Escherichia coli.

The strain Escherichia coli JRG26, which has a defect in the lipoic acid biosynthesis, was cultivated in the presence of R-lipoic acid, S-lipoic acid, RS-dithiolane-3-caproic acid, RS-bisnorlipoic acid, and RS-tetranorlipoic acid, respectively. With the exception of the last compound the strain was able to grow with all these substances. R-lipoic acid was the most efficient factor, concentrations of 10 ng/l were sufficient to support growth of the cells, while 10(4)-fold to 10(7)-fold higher concentrations were necessary for the other compounds. The specific catalytic activity of the pyruvate dehydrogenase complex isolated from the cells grown on RS-dithiolane-3-caproic acid was only slightly lower than from cells grown on R-lipoic acid. With RS bisnorlipoic acid the specific activity was one third compared to that of the native enzyme complex. The incorporation of the RS-bisnorlipoic acid into the pyruvate dehydrogenase could directly be demonstrated by polyclonal antibodies directed against R-lipoic acid and RS-bisnorlipoic acid, both conjugated to BSA. Western blot analysis showed that the antibodies against the R-lipoic acid reacted specifically with the E2 component of pyruvate dehydrogenase complex purified from cells grown on this factor, while antibodies against RS-bisnorlipoic acid reacted with the enzyme complex isolated from cells grown in the presence of this compound.

Acetyltransferases↗
PubMed1991

Disruption and mutagenesis of the Saccharomyces cerevisiae PDX1 gene encoding the protein X component of the pyruvate dehydrogenase complex.

Disruption of the PDX1 gene encoding the protein X component of the mitochondrial pyruvate dehydrogenase (PDH) complex in Saccharomyces cerevisiae did not affect viability of the cells. However, extracts of mitochondria from the mutant, in contrast to extracts of wild-type mitochondria, did not catalyze a CoA- and NAD(+)-linked oxidation of pyruvate. The PDH complex isolated from the mutant cells contained pyruvate dehydrogenase (E1 alpha + E1 beta) and dihydrolipoamide acetyltransferase (E2) but lacked protein X and dihydrolipoamide dehydrogenase (E3). Mutant cells transformed with the gene for protein X on a unit-copy plasmid produced a PDH complex that contained protein X and E3, as well as E1 alpha, E1 beta, and E2, and exhibited overall activity similar to that of the wild-type PDH complex. These observations indicate that protein X is not involved in assembly of the E2 core nor is it an integral part of the E2 core. Rather, protein X apparently plays a structural role in the PDH complex; i.e., it binds and positions E3 to the E2 core, and this specific binding is essential for a functional PDH complex. Additional evidence for this conclusion was obtained with deletion mutations. Deletion of most of the lipoyl domain (residues 6-80) of protein X had little effect on the overall activity of the PDH complex. This observation indicates that the lipoyl domain, and its covalently bound lipoyl moiety, is not essential for protein X function. However, deletion of the putative subunit binding domain (residues approximately 144-180) of protein X resulted in loss of high-affinity binding of E3 and concomitant loss of overall activity of the PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

Nucleotide sequence of a cDNA encoding the lipoate acetyl transferase (E2) of human heart pyruvate dehydrogenase complex differs from that of human placenta.

Tissue specific isoforms of an enzyme autoantigen were sought in an attempt to explain a possible disease-associated translocation of the enzyme. A human heart cDNA clone (0.66 kb) coding for part of the lipoate acetyl transferase component of pyruvate dehydrogenase complex, recently identified as one of the major autoantigens of primary biliary cirrhosis was isolated. The cloned cDNA corresponded to nucleotides 1545-2201 of a previously published placental sequence, but showed some differences which give rise to differences in the inferred amino acid sequences of proteins. This may indicate the existence of tissue-specific isoforms of the lipoate acetyl transferase component of pyruvate dehydrogenase complex coded for by a multi-gene family.

Acetyltransferases↗
PubMed1988

Mobile sequences in the pyruvate dehydrogenase complex, the E2 component, the catalytic domain and the 2-oxoglutarate dehydrogenase complex of Azotobacter vinelandii, as detected by 600 MHz 1H-NMR spectroscopy.

600 MHz 1H-NMR spectroscopy demonstrates that the pyruvate dehydrogenase complex of Azotobacter vinelandii contains regions of the polypeptide chain with intramolecular mobility. This mobility is located in the E2 component and can probably be ascribed to alanine-proline-rich regions that link the lipoyl subdomains to each other as well as to the E1 and E3 binding domain. In the catalytic domain of E2, which is thought to form a compact, rigid core, also conformational flexibility is observed. It is conceivable that the N-terminal region of the catalytic domain, which contains many alanine residues, is responsible for the observed mobility. In the low-field region of the 1H-NMR spectrum of E2 specific resonances are found, which can be ascribed to mobile phenylalanine, histidine and/or tyrosine residues which are located in the E1 and E3 binding domain that links the lipoyl domain to the catalytic domain. In the 1H-NMR spectrum of the intact complex, these resonances cannot be observed, indicating a decreased mobility of the E1 and E3 binding domain.

Acetyltransferases↗
PubMedDate not supplied

Changes in some properties of the aurochs heart pyruvate dehydrogenase complex induced by its partial phosphorylation.

Comparative studies on the properties of the dephosphorylated and partially phosphorylated (to 35% activity reduction) pyruvate dehydrogenase complex (PDC) from aurochs heart muscle have been made. Data have been obtained indicating that the partial phosphorylation of PDC abolishes the kinetic attributes of a positive cooperativity of the pyruvate binding sites (nH = 1.5) featuring at low substrate concentrations. In addition, the partially phosphorylated PDC is inactivated slower at 50 degrees C.

Animals↗
PubMed1987

Pyruvate dehydrogenase complex deficiency as a cause of subacute necrotizing encephalopathy (Leigh disease).

Leigh disease is a disorder with great clinical variability and for which diverse biochemical causes have been proposed. Clarification requires rigorous correlation of biochemical abnormalities with strict morphologic diagnosis; such an unambiguous association is the subject of this report. A patient with well-documented clinical and biochemical pyruvate dehydrogenase complex deficiency is shown on postmortem examination to have the specific CNS pathology of Leigh disease. These findings, considered together with the aggregate data in the literature, suggest strongly that pyruvate dehydrogenase complex deficiency is the basic defect in a subgroup of patients with Leigh disease.

Acidosis, Lactic↗