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PubMed1992

Leigh syndrome associated with a deficiency of the pyruvate dehydrogenase complex: results of treatment with a ketogenic diet.

A one-year-old boy suffering from intermittent lactic acidosis, muscular hypotonia, horizontal gaze paralysis and spasticity in both legs had low activity of the pyruvate dehydrogenase complex associated with low amounts of immunoreactive E 1 alpha and E 1 beta. Leigh syndrome was diagnosed on the basis of the clinical and biochemical abnormalities and the typical lesions observed on MRI of the brain. Treatment with a ketogenic diet was associated with clinical and biochemical amelioration. A striking improvement of the cerebral lesions was observed by neuro-imaging.

Brain↗
PubMed1984

Measurements of electron spin resonance with the pyruvate dehydrogenase complex from Escherichia coli. Studies on the allosteric binding site of acetyl-coenzyme A.

Binding of the feedback inhibitor acetyl-coenzyme A to the pyruvate dehydrogenase complex from Escherichia coli was studied by electron spin resonance spectroscopy with the spin-labelled acetyl-CoA analogue 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl-CoA-thioester. The spin-labelled compound binds to the pyruvate dehydrogenase component of the enzyme complex and this binding can be reversed by acetyl-CoA, while CoA has no effect. AMP and fructose 1,6-bisphosphate, which are both activators of the pyruvate dehydrogenase complex, exhibit a partial competition with the spin-labelled acetyl-CoA analogue and it could be shown that both activators act essentially by reversion of the feedback inhibition of acetyl-CoA. The binding site for these activators seems to overlap with the acetyl-CoA binding site, possibly by a common phosphate attachment point. No competition for binding to the feedback inhibition site exists with pyruvate, thiamine diphosphate, magnesium ions and with the fluorescent chromophore 8-anilino-1-naphthalene sulfonic acid. Thus, the feedback inhibition site proves to be a true allosteric regulatory site, which appears to be completely separate from the catalytic site on the pyruvate dehydrogenase component. The spin-labelled acetyl-CoA analogue binds also to the product binding site of acetyl-CoA on the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. Two binding sites per polypeptide chain with identical affinities on this enzyme component were found and the binding of the analogue can be inhibited by acetyl-CoA as well as by CoA.

Acetyl Coenzyme A↗
PubMed1980

Fluorescence polarization and energy-transfer studies on the pyruvate dehydrogenase complex of Escherichia coli.

We have attached eosin maleimide specifically to the lipoyl group of the pyruvate dehydrogenase complex isolated from Escherichia coli. Using this as the fluorescence acceptor and the intrinsic FAD of the lipoamide dehydrogenase subunit as the fluorescence donor, we confirmed previous measurements with other probes, in which it was suggested that the flavin moiety is at a substantial distance (over 4.5 nm) from the labeled lipoyl group. Since the lipoyl group must apply electrons to the FAD during the catalytic decarboxylation of pyruvate, we have investigated several potential mechanisms whereby this could happen. Movement within the complex, possibly triggered by the presence of substrate, seemed to be a strong possibility. Complex labeled with fluorophores on the accessible sulfhydryls, or on the lipoyl functions, did not give evidence of such triggering upon addition of substrate as judged by both static and dynamic fluorescence depolarization. The mobility of the subunits of labeled lipoamide dehydrogenase exceeded that expected for the total complex. Pyrene maleimide bound to the lipoyl functions also exhibited considerably faster rotations than the predicted one of the whole complex (tau c > 3 micros). This suggests that a constant movement within the complex, coupled with the rotation of the lipoyl group, may bring the active sites of the complex transiently close enough together to interact on a time scale much faster than enzyme turnover. At the same time, the lipoyl group and the active sites of the complex can spend most of their time at points which are rather distant from each other.

Chemical Phenomena↗
PubMed1986

The activity of the pyruvate dehydrogenase complex in heart muscle in the previously obese mouse model.

Obese gold thioglucose injected mice were reduced to lean control weight by food restriction. When pair fed with lean controls these animals then gained weight (were metabolically more efficient). Serum glucose was also elevated in this group (14.5 +/- 0.4 (14) vs 12.1 +/- 0.3 mmol/L, p less than 0.001). If previously obese animals were weight maintained with lean controls (by mild food restriction), serum glucose remained at control levels. The activity of the pyruvate dehydrogenase complex in heart muscle was decreased in both obese and pair fed previously obese, whilst it was similar to that of lean controls in the weight maintained previously obese and in obese mice actually dieted. In all obese and previously obese animals serum insulin was elevated. In hearts from control animals subjected to mild food restriction the pyruvate dehydrogenase complex was activated (11.53 +/- 1.80 (5) vs 3.34 +/- 0.62 (9) U/g dry weight), despite a reduced serum insulin level (42 +/- 2 vs 74 +/- 10 microU/ml, p less than 0.01). These diverse changes in the proportion of the pyruvate dehydrogenase complex in the active form and insulin levels argue for a persistent alteration in the sensitivity of the pyruvate dehydrogenase complex to insulin in obesity, as well as indicating that glucose metabolism in obese animals is altered by both body weight and diet amount.

Animals↗
PubMed1994

The nucleotide and deduced amino acid sequences of a cDNA encoding the E1 beta-subunit of the Arabidopsis thaliana mitochondrial pyruvate dehydrogenase complex.

A cDNA encoding the E1 beta subunit of the Arabidopsis thaliana mitochondrial pyruvate dehydrogenase complex was sequenced. The 1230 bp cDNA contains a 1089-base open reading frame encoding a polypeptide of 363 amino acids with a predicted molecular mass of 39,190 Da and an isoelectric point of 4.9. A 29-residue presumptive mitochondrial targeting sequence is present at the amino terminus.

Amino Acid Sequence↗
PubMed2001

Cooperation of divalent ions and thiamin diphosphate in regulation of the function of pig heart pyruvate dehydrogenase complex.

The role of Mg2+, Ca2+, and Mn2+ in regulation of purified pig heart pyruvate dehydrogenase complex (PDC) containing endogenous thiamin diphosphate (TDP) was studied. It was found that the effects of the cations depended on the presence of exogenous TDP. In the absence of added TDP, the divalent cations led to a shortening of a lag phase of the PDC reaction and a strong reduction of the Km value for pyruvate. The relative efficiency of the three types of ions are presented as follows: Mn2+>Ca2+>Mg2+. The other sources claim that in the presence of exogenous TDP, which alone strongly increased the affinity of PDC for pyruvate, any significant additional effects of the cations were not observed. However, Mg2+, Ca2+, and Mn2+ decreased the Km value for CoA in both cases, the absence and presence of exogenous TDP, in approximately a similar extent (about twofold). The affinity of PDC for NAD+ seems to be not sensitive to the presence of the divalent cations. The data obtained suggest that Mg2+, Ca2+, and Mn2+ can cooperate with TDP as positive regulatory effectors of pig heart PDC on the level of pyruvate dehydrogenase and lipoamide acetyltransferase components of the complex.

Animals↗
PubMed1986

Comparative development of the pyruvate dehydrogenase complex and citrate synthase in rat brain mitochondria.

The enzyme activity of the pyruvate dehydrogenase complex (PDHC) was measured in mitochondria prepared from developing rat brain, before and after steady-state dephosphorylation of the E1 alpha subunit. A marked increase in dephosphorylated (fully activated) PDHC activity occurred between days 10 and 15 post partum, which represented approx. 60% of the difference in fully activated PDHC activity measured in foetal and adult rat brain mitochondria. There was no detectable change in the active proportion of the enzyme during mitochondrial preparation nor any qualitative alteration in the detectable catalytic and regulatory components of the complex, which might account for developmental changes in PDHC activity. The PDHC protein content of developing rat brain mitochondria and homogenates was measured by an enzyme-linked immunoadsorbent assay. The development of PDHC protein in both fractions agreed closely with the development of the PDHC activity. The results suggest that the developmental increase in PDHC activity is due to increased synthesis of PDHC protein, which is partly a consequence of an increase in mitochondrial numbers. However, the marked increase in PDHC activity measured between days 10 and 15 post partum is mainly due to an increase in the amount of PDHC per mitochondrion. The development of citrate synthase enzyme activity and protein was measured in rat brain homogenates and mitochondria. As only a small increase in citrate synthase activity and protein was detected in mitochondria between days 10 and 15 post partum, the marked increase in PDHC protein and enzyme activity may represent specific PDHC synthesis. As several indicators of acquired neurological competence become apparent during this period, it is proposed that preferential synthesis of PDHC may be crucial to this process. The results are discussed with respect to the possible roles played by PDHC in changes of respiratory-substrate utilization and the acquisition of neurological competence occurring during the development of the brain of a non-precocial species such as the rat.

Animals↗
PubMed2001

Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases.

This review summarizes the recent developments on the regulation of human pyruvate dehydrogenase complex (PDC) by site-specific phosphorylation by four kinases. Mutagenic analysis of the three phosphorylation sites of human pyruvate dehydrogenase (E1) showed the site-independent mechanism of phosphorylation as well as site-independent dephosphorylation of the three phosphorylation sites and the importance of each phosphorylation site for the inactivation of E1. Both the negative charge and size of the group introduced at site 1 were involved in human E1 inactivation. Mechanism of inactivation of E1 was suggested to be site-specific. Phosphorylation of site 1 affected E1 interaction with the lipoyl domain of dihydrolipoamide acetyltransferase, whereas phosphorylation site 3 appeared to be closer to the thiamine pyrophosphate (TPP)-binding region affecting coenzyme interaction with human E1. Four isoenzymes of pyruvate dehydrogenase kinase (PDK) showed different specificity for the three phosphorylation sites of E1. All four PDKs phosphorylated sites 1 and 2 in PDC with different rates, and only PDK1 phosphorylated site 3. PDK2 was maximally stimulated by the reduction/acetylation of the lipoyl groups of E2. Presence of the multiple phosphorylation sites and isoenzymes of PDK is important for the tissue-specific regulation of PDC under different physiological conditions.

Acetylation↗
PubMed1995

Hepatic distribution of E2 component of pyruvate dehydrogenase complex after transplantation.

We have examined the distribution of the E2 component of pyruvate dehydrogenase complex in the liver of patients with native primary biliary cirrhosis (PBC), and after transplantation, using affinity-purified anti-E2 antibodies. In the posttransplantation group, we studied biopsy specimens from patients grafted for conditions other than PBC (n = 6) and those grafted for PBC with (n = 6) and without (n = 5) histological features suggestive of PBC recurrence. Features suggestive of PBC recurrence included portal tract granulomas, bile duct damage, ductopenia, and lymphoid aggregates. In the native liver from patients with PBC, there was increased staining of E2 on the biliary epithelial cells compared with hepatocytes, as previously described. However, in liver biopsy specimens from patients after transplantation, the pattern of staining of E2 was similar to that of normal, control liver in all three groups studied. These findings suggest that E2 overexpression on bile duct cells may not be important in the perpetuation of the bile duct damage in PCB, that expression in the allograft may be modified by immunosuppression, or that PBC does not recur in the allograft.

Biopsy↗
PubMed2006

A Korean female patient with thiamine-responsive pyruvate dehydrogenase complex deficiency due to a novel point mutation (Y161C)in the PDHA1 gene.

Pyruvate dehydrogenase complex (PDHC) deficiency is mostly due to mutations in the X-linked E1alpha subunit gene (PDHA1). Some of the patients with PDHC deficiency showed clinical improvements with thiamine treatment. We report the results of biochemical and molecular analysis in a female patient with lactic acidemia. The PDHC activity was assayed at different concentrations of thiamine pyrophosphate (TPP). The PDHC activity showed null activity at low TPP concentration (1 x 10(-3) mM), but significantly increased at a high TPP concentration (1 mM). Sequencing analysis of PDHA1 gene of the patient revealed a substitution of cysteine for tyrosine at position 161 (Y161C). Thiamine treatment resulted in reduction of the patient's serum lactate concentration and dramatic clinical improvement. Biochemical, molecular, and clinical data suggest that this patient has a thiamine-responsive PDHC deficiency due to a novel mutation, Y161C. Therefore, to detect the thiamine responsiveness it is necessary to measure activities of PDHC not only at high but also at low concentration of TPP.

Cells, Cultured↗
PubMed1989

Antimitochondrial autoantibodies in primary biliary cirrhosis recognize cross-reactive epitope(s) on protein X and dihydrolipoamide acetyltransferase of pyruvate dehydrogenase complex.

Antimitochondrial autoantibodies are characteristically present in sera of patients with primary biliary cirrhosis. The antimitochondrial autoantibodies recognize four major antigens from beef heart mitochondria at relative molecular weights of 74, 56, 52 and 48 kD. In the present study, we report that the 56 kD antigen is the protein X of pyruvate dehydrogenase complex and that it possesses cross-reactive antimitochondrial autoantibody epitope(s) with the 74 kD antigen, the acetyltransferase (E2) of the pyruvate dehydrogenase complex. This was demonstrated by comparing the specificities of primary biliary cirrhosis sera with a protein X-specific rabbit antiserum and by absorbing primary biliary cirrhosis sera with recombinant pyruvate dehydrogenase-E2 fusion protein. In the two-dimensional gel analysis, primary biliary cirrhosis sera and protein X-specific rabbit antiserum reacted to the same two isoelectric point polypeptides at 56 kD molecular weight. The absorption of primary biliary cirrhosis sera with the human recombinant pyruvate dehydrogenase-E2 removed reactivity toward both the 74 and 56 kD antigens. Furthermore, analysis of 82 antimitochondrial autoantibody-positive primary biliary cirrhosis sera by immunoblotting did not reveal any sera which reacted solely against either the 74 or 56 kD antigen. Finally, primary biliary cirrhosis sera recognized protein X from human, bovine and porcine sources but not protein X from rat or mouse origin. The identification of protein X as another major target of the autoimmune response in primary biliary cirrhosis suggests that the pyruvate dehydrogenase complex may have a central role in the induction of this enigmatic disease.

Acetyltransferases↗
PubMed1995

Interaction of alpha-lipoic acid enantiomers and homologues with the enzyme components of the mammalian pyruvate dehydrogenase complex.

Lipoic acid (alpha-lipoic acid, thioctic acid) is applied as a therapeutic agent in various diseases accompanied by polyneuropathia such as diabetes mellitus. The stereoselectivity and specificity of lipoic acid for the pyruvate dehydrogenase complex and its component enzymes from different sources has been studied. The dihydrolipoamide dehydrogenase component from pig heart has a clear preference for R-lipoic acid, a substrate which reacts 24 times faster than the S-enantiomer. Selectivity is more at the stage of the catalytic reaction than of binding. The Michaelis constants of both enantiomers are comparable (Km = 3.7 and 5.5 mM for R- and S-lipoic acid, respectively) and the S-enantiomer inhibits the R-lipoic acid dependent reaction with an inhibition constant similar to its Michaelis constant. When three lipoic acid homologues were tested, RS-1,2-dithiolane-3-caproic acid was one carbon atom longer than lipoic acid, while RS-bisnorlipoic acid and RS-tetranorlipoic acid were two and four carbon atoms shorter, respectively. All are poor substrates but bind to and inhibit the enzyme with an affinity similar to that of S-lipoic acid. No essential differences with respect to its reaction with lipoic acid enantiomers and homologues exist between free and complex-bound dihydrolipoamide dehydrogenase. Dihydrolipoamide dehydrogenase from human renal carcinoma has a higher Michaelis constant for R-lipoic acid (Km = 18 mM) and does not accept the S-enantiomer as a substrate. Both enantiomers of lipoic acid are inhibitors of the overall reaction of the bovine pyruvate dehydrogenase complex, but stimulate the respective enzyme complexes from rat as well as from Escherichia coli. The S-enantiomer is the stronger inhibitor, the R-enantiomer the better activator. The two enantiomers have no influence on the partial reaction of the bovine pyruvate dehydrogenase component, but do inhibit this enzyme component from rat kidney. The implications of these results are discussed.

Animals↗
PubMed1984

The effect of ischaemia on the activity of pyruvate dehydrogenase complex in rat heart.

The effect of ischaemia on the concentration of active pyruvate dehydrogenase complex has been investigated in glucose perfused hearts of normal rats fed a normal diet or a high fat diet or starved for 48 h; and in hearts from alloxan-diabetic rats. Global ischaemia induced by low flow (approx. 1 ml/min) lowered the concentration of active complex under most of the experimental conditions employed. Parallel studies showed that anoxia and K+ arrest of the heart had effects similar to that of ischaemia and suggested that hypoxia and decreased mechanical activity of the heart may be responsible for effects of low flow ischaemia. Evidence is reviewed that the effects of low flow ischaemia, K+ arrest and anoxia may be mediated through activation of pyruvate dehydrogenase kinase by increased reduction of mitochondrial NAD+. In hearts of normal rats on a normal diet, global ischaemia induced by zero flow and regional ischaemia induced by coronary artery ligation increased the concentration of active complex. Evidence is given that this may result from a combination of anoxia and acidosis. In aerobic perfusions at 60 mmHg, concentrations of active complex were ranked in the order: normal diet greater than high fat diet greater than 48 h starved greater than alloxan diabetic. This order was maintained when the concentration of active complex was increased by perfusion at 120 mmHg or lowered by global ischaemia induced by zero flow.

Adenosine Triphosphate↗
PubMed2001

Heterogeneous response of antimitochondrial autoantibodies and bile duct apical staining monoclonal antibodies to pyruvate dehydrogenase complex E2: the molecule versus the mimic.

The 2-oxo-acid dehydrogenase complexes and, in particular, the E2 component of the pyruvate dehydrogenase complex (PDC) are the target of antimitochondrial antibodies (AMA). More than 95% of primary biliary cirrhosis (PBC) patients have detectable levels of autoantibodies to PDC-E2 and in general these react with a region of the molecule that contains the prosthetic group lipoic acid (LA). LA is vital to the function of the enzyme, although there is conflicting evidence as to whether its presence is required for PDC-E2 recognition by AMA. Some, but not all, monoclonal antibodies (mAbs) to PDC-E2 produce an intense staining pattern at the apical surface of bile duct epithelial cells (BEC) in patients with PBC, and it has been argued that the molecule at the apical surface of PBC bile duct cells is a modified form of PDC-E2 or a cross-reactive molecule, acting as a molecular mimic. Herein, we characterize the epitopes recognized by 4 anti-PDC-E2 mAbs that give apical staining patterns (3 mouse and 1 human). In particular, by using a combination of recombinant antigens, competitive inhibition assays, and a unique peptide-on-bead assay, we determined that these apically staining mAbs recognize 3 or 4 distinct epitopes on PDC-E2. More importantly, this suggests that a portion spanning the entire inner lipoyl domain of PDC-E2 can be found at the BEC apical surface. In addition, competition assays with patient sera and a PDC-E2-specific mAb showed significant epitope overlap with only 1 of the 3 mouse mAbs and showed a differential response to the peptide bound to beads. These findings further highlight the heterogeneous response of patient autoantibodies to the inner lipoyl domain of PDC-E2.

Amino Acid Sequence↗
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↗
PubMedDate not supplied

Configuration of interdomain linkers in pyruvate dehydrogenase complex of Escherichia coli as determined by cryoelectron microscopy.

The dihydrolipoyl transacetylase (E2p) component of the pyruvate dehydrogenase complex (PDC) of Escherichia coli is a multidomain polypeptide comprising a catalytic domain, a domain that binds dihydrolipoyl dehydrogenase (E3-binding domain), and three domains containing lipoic acid (lipoyl domains). In PDC 24 subunits of E2p associate by means of interactions involving the catalytic domains to form the structural core of PDC. From cryoelectron microscopy and computer image analysis of frozen-hydrated isolated E2p cores it appears that the lipoyl domains are located peripherally about the core complex and do not assume fixed positions. To further test this interpretation the visibility of the lipoyl domains in electron micrographs was enhanced by specifically biotinylating the lipoic acids and labeling them with streptavidin. In agreement with the studies of native, unlabeled E2p cores, cryoelectron microscopy of the streptavidin-labeled E2p cores showed that the lipoic acid moieties are capable of extending approximately 13 nm from the surface of the core. Localization of the E3-binding domains was accomplished by cryoelectron microscopy of E2p-E3 subcomplexes prepared by reconstitution in vitro. Frequently an apparent gap of several nanometers separated the bound E3 from the surface of the core. The third component of PDC, pyruvate dehydrogenase (E1p), appeared to bind to the E2p core in a manner similar to that observed for E3. These results support a structural model of the E2p core in which the catalytic, E3-binding, and three lipoyl domains are interconnected by linker sequences that assume extended and flexible conformations.

Bacterial Proteins↗
PubMed1990

Changes in pyruvate dehydrogenase complex (PDHc) activity and [3H]QNB-receptor binding in rat brain subsequent to intracerebroventricular injection of bromopyruvate.

Pyruvate dehydrogenase complex (PDHc), a link between carbohydrate and acetylcholine metabolism, is a regulatory enzyme for glucose and neurotransmitter metabolism in the brain and is reduced in Alzheimer-diseased brain. To study functional consequences of an inhibition of PDHc on muscarinic receptor binding, bromopyruvate, a suicide inhibitor od PDHc, was injected intracerebroventricularly (icv) in rats. Bromopyruvate caused a reduction of PDHc activity in the 3 brain regions examined, however, reaching significance only in the cerebral cortex and the hippocampus and not in the striatum, 24 h after injection. 3, 6, and 12 weeks later, there was a normalization or transiently increased activity, respectively, of PDHc in these brain regions. No changes in concentrations of energy-rich phosphates could be demonstrated in the cerebral cortex 12 weeks after brompyruvate injection. The number of muscarinic receptors was significantly reduced in the cerebral cortex 12 weeks after injection. The data indicate that a transient reduction of brain PDHc activity in vivo is associated with a long-lasting reduction in muscarinic cholinergic receptors. Because comparable changes of PDHc and muscarinic receptors are found in dementia of Alzhemier type, the model of bromopyruvate inhibition of PDHc in rats is suggested to be useful for experimental dementia research.

Alzheimer Disease↗