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PubMed2006

Therapeutic potential of dichloroacetate for pyruvate dehydrogenase complex deficiency.

We reviewed the use of oral dichloroacetate (DCA) in the treatment of children with congenital lactic acidosis caused by mutations in the pyruvate dehydrogenase complex (PDC). The case histories of 46 subjects were analyzed with regard to diagnosis, clinical presentation and response to DCA. DCA decreased blood and cerebrospinal fluid lactate concentrations, and was generally well tolerated. DCA may be particularly effective in children with PDC deficiency by stimulating residual enzyme activity and, consequently, cellular energy metabolism. A controlled trial is needed to determine the definitive role of DCA in the management of this devastating disease.

Acidosis, Lactic↗
PubMed1983

Decreased pyruvate dehydrogenase complex activity in Huntington and Alzheimer brain.

The activity of the pyruvate dehydrogenase complex (PDHC) was reduced in affected areas of brain from patients with Huntington disease (caudate, putamen) and Alzheimer disease (frontal cortex) where choline acetyltransferase (CAT) activity was low. PDHC was also deficient in an area (Huntington hippocampus) where CAT was not significantly reduced. The activity of fumarase, an inner mitochondrial marker, was normal in all areas examined. The activities of PDHC and CAT correlated well in caudate, putamen, and amygdala but not in hippocampus or frontal cortex. Both total activity and activation of PDHC were below normal in fibroblasts from 4 patients with C-21 trisomy Down syndrome, who are at very high risk to develop Alzheimer disease. However, no abnormality of PDHC was detected in Huntington or Alzheimer fibroblasts. Deficiency of PDHC may play a role in the pathophysiology of Huntington and Alzheimer diseases, although it does not appear to be a primary defect. Loss of tissue oxidative capacity may relate to the reduction in cerebral metabolic rate and blood flow which are characteristic of many dementing illnesses.

Alzheimer Disease↗
PubMedDate not supplied

Pyruvate dehydrogenase complex: metabolic link to ischemic brain injury and target of oxidative stress.

The mammalian pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme complex (greater than 7 million Daltons) that catalyzes the oxidative decarboxylation of pyruvate to form acetyl CoA, nicotinamide adenine dinucleotide (the reduced form, NADH), and CO(2). This reaction constitutes the bridge between anaerobic and aerobic cerebral energy metabolism. PDHC enzyme activity and immunoreactivity are lost in selectively vulnerable neurons after cerebral ischemia and reperfusion. Evidence from experiments carried out in vitro suggests that reperfusion-dependent loss of activity is caused by oxidative protein modifications. Impaired enzyme activity may explain the reduced cerebral glucose and oxygen consumption that occurs after cerebral ischemia. This hypothesis is supported by the hyperoxidation of mitochondrial electron transport chain components and NAD(H) that occurs during reperfusion, indicating that NADH production, rather than utilization, is rate limiting. Additional support comes from the findings that immediate postischemic administration of acetyl-L-carnitine both reduces brain lactate/pyruvate ratios and improves neurologic outcome after cardiac arrest in animals. As acetyl-L-carnitine is converted to acetyl CoA, the product of the PDHC reaction, it follows that impaired production of NADH is due to reduced activity of either PDHC or one or more steps in glycolysis. Impaired cerebral energy metabolism and PDHC activity are associated also with neurodegenerative disorders including Alzheimer's disease and Wernicke-Korsakoff syndrome, suggesting that this enzyme is an important link in the pathophysiology of both acute brain injury and chronic neurodegeneration.

Animals↗
PubMed1989

Pyruvate dehydrogenase complex deficiency: biochemical and immunoblot analysis of cultured skin fibroblasts.

Cultured skin fibroblasts were obtained from 11 children with lactic acidemia and neurological disturbances. The residual activities of pyruvate dehydrogenase complex were 9 to 45% of control values in all specimens. Immunoblot analysis of mitochondrial proteins using polyclonal antibodies against the alpha and beta subunits of the first component (E1) of the pyruvate dehydrogenase complex revealed markedly decreased amounts of cross-reacting material in 4 boys who died in infancy. Two of the boys were half brothers related through a common mother. A fifth boy had an alteration of the electrophoretic mobility of the E1 alpha subunit and normal E1 beta subunit abundance. The remaining 6 patients (2 boys and 4 girls) had normal findings on Western blot assay, and all 11 patients had normal E2 and E3 patterns. These findings suggest that the E1 alpha subunit gene represents a genetically vulnerable site on the X chromosome. Decreased abundance of E1 components appears to be associated with death in infancy. A normal Western blot analysis is compatible with long-term survival despite decreased catalytic activity of the pyruvate dehydrogenase complex.

Cells, Cultured↗
PubMed1990

Expression in Escherichia coli of a sub-gene encoding the lipoyl domain of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.

A sub-gene encoding the lipoyl domain (residues 1-85) of the lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. Approx. 80% of the domain was unlipoylated but most of the remainder was correctly lipoylated on Lys-42 and could be reductively acetylated by the B stearothermophilus enzyme complex. A small proportion (approx. 4%) of the domain carried an aberrant substituent, possibly an octanoyl group, on Lys-42. The 400 MHz 1H NMR spectra of the lipoylated and unlipoylated domains were essentially identical and closely resembled that of the native lipoyl domain.

Acetyltransferases↗
PubMed1980

Bovine kidney pyruvate dehydrogenase complex. Limited proteolysis and molecular structure of the lipoate acetyltransferase component.

1. Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated by elastase in a similar manner as described earlier for papain. The core component, lipoate acetyltransferase, is cleaved by elastase into an active fragment (Mr 26000) and a fragment with apparent Mr of 45000 as analyzed by dodecylsulfate gel electrophoresis. Due to the fragmentation of the core, the enzyme complex is disassembled into its component enzymes which retain their complete enzymatic activities as assayed separately. 2. A different mechanism was found for the inactivation of pyruvate dehydrogenase complex with trypsin and some other proteases (chymotrypsin, clostripain). In these cases, the pyruvate dehydrogenase component is inactivated rapidly by limited proteolysis. More slowly, the enzyme complex is disassembled simultaneously with fragmentation of the lipoate acetyltransferase which again results in an active fragment of Mr 26000 and another fragment of apparent Mr 45000. Upon prolonged proteolysis, the latter fragment is cleaved further to give products of Mr 36000 or lower. 3. The enzyme-bound lipoyl residues of the pyruvate dehydrogenase complex have been labelled covalently by incubation with [2-14C]pyruvate. After treatment of this [14C]acetyl-enzyme with papain, elastase, or trypsin, radioactivity was associated exclusively with the 45000-Mr and 36000-Mr fragments but not with the active 26000-Mr fragment. 4. It is concluded that the bovine kidney lipoate acetyltransferase core is composed of 60 subunits each consisting of two dissimilar folding domains. One of these contains the intersubunit binding sites as well as the active center for transacylation whereas the other possesses the enzyme-bound lipoyl residues.

Acetyltransferases↗
PubMedDate not supplied

[Pyruvate dehydrogenase complex in the rabbit adrenal glands: activation by administration of ACTH].

The pyruvate dehydrogenase complex (PDC) from the rabbit adrenal mitochondria was partially purified (18-fold) and its main kinetic parameters were determined: Michaelis' constants for pyruvate, CoA and NAD are 16, 10 and 140, respectively. The inhibitor constant for acetyl-CoA is 25 microM. Phosphate ions augment the PDC affinity for the substrate. After ACTH administration to rabbits the active PDC form in the adrenals rises simultaneously with the PDC phosphatase activity growth. The data obtained are indicative of the adrenal PDC involvement in mobilizing metabolism to provide intensive steroidogenesis.

11-Hydroxycorticosteroids↗
PubMed1988

The dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Molecular cloning and sequence analysis.

The gene encoding the dihydrolipoyltransacetylase component (E2) of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been cloned in Escherichia coli. A plasmid containing a 2.8-kbp insert of A. vinelandii chromosomal DNA was obtained and its nucleotide sequence determined. The gene comprises 1911 base pairs, 637 codons excluding the initiation codon GUG and stop codon UGA. It is preceded by the gene encoding the pyruvate dehydrogenase component (E1) of pyruvate dehydrogenase complex and by an intercistronic region of 11 base pairs containing a good ribosome binding site. The gene is followed downstream by a strong terminating sequence. The relative molecular mass (64913), amino acid composition and N-terminal sequence are in good agreement with information obtained from studies on the purified enzyme. Approximately the first half of the gene codes for the lipoyl domain. Three very homologous sequences are present, which are translated in three almost identical units, alternated with non-homologous regions which are very rich in alanyl and prolyl residues. The N-terminus of the catalytic domain is sited at residue 381. Between the lipoyl domain and the catalytic domain, a region of about 50 residues is found containing many charged amino acid residues. This region is characterized as a hinge region and is involved in the binding of the pyruvate dehydrogenase and lipoamide dehydrogenase components. The homology with the dihydrolipoyltransacetylase from E. coli is high: 50% amino acid residues are identical.

Acetyltransferases↗
PubMed1987

A mutant pyruvate dehydrogenase complex of Escherichia coli deleted in the (alanine + proline)-rich region of the acetyltransferase component.

The acetyltransferase chains of the pyruvate dehydrogenase complex of Escherichia coli contain conformationally mobile (alanine + proline)-rich segments that link the lipoyl domains to each other and to the subunit-binding and catalytic domain, and facilitate the intramolecular coupling of active sites in the complex. A deletion of 12 of the 32 residues of the (Ala + Pro)-rich segment of an acetyltransferase containing only one lipoyl domain was made by deleting the corresponding segment of the aceF gene. A pyruvate dehydrogenase complex was still produced and the catalytic activity of the restructured complex, including active-site coupling, was not detectably impaired.

Acetyltransferases↗
PubMed1995

Dissociation and reassembly of the dihydrolipoyl transacetylase component of the bovine heart pyruvate dehydrogenase complex.

The catalytic activity and the state of aggregation of the dihydrolipoyl transacetylase-lipoamide dehydrogenase binding protein (E2-E3BP) subcomplex of the bovine heart pyruvate dehydrogenase multienzyme complex were investigated. Treatment of E2-E3BP with the chaotropic salts GndnCl or KSCN led to a rapid decrease in transacetylase activity which was accompanied by a loss of the native quaternary structure, as indicated by changes in the sedimentation properties of the E2-E3BP subcomplex. Reassembly or refolding of dissociated E2-E3BP was achieved for the GndnCl-treated subcomplex using a defined protocol. This reassembly procedure effectively excluded all E3BP from the reassembled oligomeric transacetylase. The reassembled oligomeric E2, free of E3BP, was unable to reconstitute the overall activity of the complex following incubation with pyruvate dehydrogenase (E1) and lipoamide dehydrogenase (E3). In binding studies using radiolabeled components it was demonstrated that the reassembled transacetylase, while retaining its capacity for reductive acetylation and its ability to bind E1, lost its ability to bind E3. The evidence presented in this study indicates that the strong association of E3BP with E2 facilitates the binding of E3, the lipoamide dehydrogenase component, and therefore may have an important role in the assembly and ultimately the catalytic activity of the pyruvate dehydrogenase multienzyme complex.

Acetyltransferases↗
PubMed1984

Regulation of pyruvate dehydrogenase complex in ischemic rat heart.

The effect of flow-induced ischemia on the rate of pyruvate decarboxylation and the activation state of the pyruvate dehydrogenase multienzyme complex was investigated in the isolated, perfused rat heart. Pyruvate dehydrogenase activity in the heart decreased significantly during flow-induced ischemia and was a function of changes in the activation state (i.e., active/total activity) of the enzyme complex. In the absence of pyruvate, the activation state of pyruvate dehydrogenase decreased from nearly 100% active at the normal flow rate (10 ml/min) to 20% active as the flow was reduced to 0.5 ml/min. At high pyruvate levels (5 mM), the activation state increased from nearly 70% active at control flow rates to 100% active during ischemia. At an intermediate pyruvate concentration (0.5 mM), the enzyme complex was maintained at a relatively low activation state (30-35% active) throughout the range of flow rates tested. Ischemia caused elevated perfusate lactate concentrations only when the flow rates were less than 5.0 ml/min. The activation state of the pyruvate dehydrogenase complex in hearts perfused with glucose was also decreased during ischemia.

Adenine Nucleotides↗
PubMed1987

The human pyruvate dehydrogenase complex. Isolation of cDNA clones for the E1 alpha subunit, sequence analysis, and characterization of the mRNA.

cDNA clones corresponding to the entire length of mRNA for the alpha subunit of human pyruvate dehydrogenase (EC 1.2.4.1), the E1 component of the pyruvate dehydrogenase complex, have been isolated from liver cDNA libraries. Two classes of cDNA clones were obtained and these correspond to two forms of pyruvate dehydrogenase E1 alpha mRNA. Both mRNA species have been demonstrated in a variety of human tissues and cultured fibroblasts. The cDNA sequence has been determined and, from it, the protein sequence of the human E1 alpha subunit was deduced. The protein is synthesized with a typical mitochondrial import leader sequence and the peptide bond at which this sequence is cleaved after transport into the mitochondrion has been determined by direct amino acid sequencing of the mature E1 alpha subunit. The human pyruvate dehydrogenase E1 alpha subunit contains identical phosphorylation sites to those found in the corresponding porcine protein. Preliminary studies of pyruvate dehydrogenase E1 alpha mRNA in cultured fibroblasts from patients with severe pyruvate dehydrogenase deficiency have revealed considerable heterogeneity as would be expected from protein studies.

Amino Acid Sequence↗
PubMed1994

Sequential 1H and 15N nuclear magnetic resonance assignments and secondary structure of the N-terminal lipoyl domain of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii.

The N-terminal lipoyl domain (79 residues) of the transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been sub-cloned and produced in Escherichia coli. Over-expression exceeds the capacity of E. coli cells to lipoylate all expressed lipoyl domain, but addition of lipoic acid to the growth medium results in expression of fully lipoylated domain. A two-dimensional homo- and heteronuclear NMR study of the lipoyl domain has resulted in sequential 1H and 15N resonance assignments of the unlipoylated form of the protein. Small differences in chemical shift values for protons of residues in the vicinity of the lipoyl-lysine residue are observed for the lipoylated form of the domain, suggesting that the conformation of the lipoyl domain is not altered significantly by the coupled cofactor. From nuclear Overhauser effects, backbone coupling constants and slowly exchanging amide protons, two antiparallel beta-sheets, each containing four strands, were identified. The lipoyl-lysine residue is exposed to the solvent and located in a type-I turn between two strands. The N- and C-terminal residues of the folded chain are close together in the other sheet. Preliminary data on the relative three-dimensional orientation of the two beta-sheets are presented. Comparison with the solution structure of the lipoyl domain of the Bacillus stearothermophilous pyruvate dehydrogenase complex shows resemblance to a large extent, despite the sequence identity of 31%.

Acetyltransferases↗
PubMed1999

Metabolic acidosis inhibits pyruvate oxidation in chick liver by decreasing activity of pyruvate dehydrogenase complex.

Replacement of drinking water with NH4Cl (1.5%) solution significantly reduced blood pH on the 2nd d in chicks and thereafter. Concomitant with this reduction, oxidation rate of state 3 with pyruvate in liver mitochondria was also decreased in acidotic animals when compared with control animals. No significant differences between the two groups were observed in the state 4 oxidation at any feeding period. The ADP/O ratio did not appear to be affected by the treatment. The successive experiments of gavage-feeding for 4 d were also employed to ensure an equivalent intake of diet and the amount of NH4Cl given. As a result, the higher the NH4Cl provided, the lower the oxidation rate of state 3 with pyruvate in liver mitochondria, and the actual activity of pyruvate dehydrogenase complex, as expressed as units of produced CO2 per g wet weight of liver, which were accompanied by the lower pH in blood. This study provides the first evidence for a critical role of pyruvate dehydrogenase complex in the regulation of pyruvate catabolism in the liver from acidotic chicks induced by NH4Cl.

Acidosis↗
PubMed2005

The malaria parasite Plasmodium falciparum has only one pyruvate dehydrogenase complex, which is located in the apicoplast.

The relict plastid (apicoplast) of apicomplexan parasites synthesizes fatty acids and is a promising drug target. In plant plastids, a pyruvate dehydrogenase complex (PDH) converts pyruvate into acetyl-CoA, the major fatty acid precursor, whereas a second, distinct PDH fuels the tricarboxylic acid cycle in the mitochondria. In contrast, the presence of genes encoding PDH and related enzyme complexes in the genomes of five Plasmodium species and of Toxoplasma gondii indicate that these parasites contain only one single PDH. PDH complexes are comprised of four subunits (E1alpha, E1beta, E2, E3), and we confirmed four genes encoding a complete PDH in Plasmodium falciparum through sequencing of cDNA clones. In apicomplexan parasites, many nuclear-encoded proteins are targeted to the apicoplast courtesy of two-part N-terminal leader sequences, and the presence of such N-terminal sequences on all four PDH subunits as well as phylogenetic analyses strongly suggest that the P. falciparum PDH is located in the apicoplast. Fusion of the two-part leader sequences from the E1alpha and E2 genes to green fluorescent protein experimentally confirmed apicoplast targeting. Western blot analysis provided evidence for the expression of the E1alpha and E1beta PDH subunits in blood-stage malaria parasites. The recombinantly expressed catalytic domain of the PDH subunit E2 showed high enzymatic activity in vitro indicating that pyruvate is converted to acetyl-CoA in the apicoplast, possibly for use in fatty acid biosynthesis.

Acetyl Coenzyme A↗
PubMed1992

Additional binding sites for the pyruvate dehydrogenase kinase but not for protein X in the assembled core of the mammalian pyruvate dehydrogenase complex: binding region for the kinase.

A standard resolution of the bovine kidney pyruvate dehydrogenase complex yields a subcomplex composed of approximately 60 dihydrolipoyl transacetylase (E2) subunits, approximately 6 protein X subunits, and approximately 2 pyruvate dehydrogenase kinase heterodimers (KcKb). Using a preparation of resolved kinase in which Kc much greater than Kb, E2-X-KcKb subcomplex additionally bound at least 15 catalytic subunits of the kinase (Kc) and a much lower level of Kb. The binding of Kc to E2 greatly enhanced kinase activity even at high levels of bound kinase. Free protein X, functional in binding the E3 component, did not bind to E2-X-KcKb subcomplex. This pattern of binding Kc but not protein X was unchanged either with a preparation of E2 oligomer greatly reduced in protein X or with subcomplex from which the lipoyl domain of protein X was selectively removed. The bound inner domain of protein X associated with the latter subcomplex did not exchange with free protein X. These data support the conclusion that E2 subunits bind the Kc subunit of the kinase and suggest that the binding of the inner domain of protein X to the inner domain of the transacetylase occurs during the assembly of the oligomeric core. Selective release of a fragment of E2 subunits that contain the lipoyl domains (E2L fragment) releases the kinase (M. Rahmatullah et al., 1990, J. Biol. Chem. 265, 14,512-14,517). Sucrose gradient centrifugation yielded an E2L-kinase fraction with an increased ratio of the kinase to E2L fragment. A monoclonal antibody specific for E2L was attached to a gel matrix. Binding of E2L fragment also led to specific binding of the kinase. Extensive washing did not reduce the level of bound kinase. Thus, the kinase is tightly bound by the lipoyl domain region of E2.

Acetyltransferases↗
PubMed1995

Dissociation and unfolding of the pyruvate dehydrogenase complex by guanidinium chloride.

The effect of guanidinium chloride (GdnHCl) on the pyruvate dehydrogenase complex (PDC) from bovine heart and its constituent enzymes has been studied. The overall activity of the complex is lost reversibly at low levels of GdnHCl (0.2 M) which cause 40-50% inactivation but no loss of overall secondary or tertiary structures of the individual enzymes; the inactivation of the complex is shown to be caused by dissociation of the E1 and E3 components from the E2/X core assembly. This provides an improved procedure for controlled dissociation of the complex and efficient recovery of its component enzymes in their native states. Higher concentrations of GdnHCl (up to 4 M) lead to the unfolding and irreversible inactivation of the separate enzymes of the complex with the E2/X core proving the most resistant to GdnHCl-induced unfolding. Neither the 60-meric E2/X core assembly nor the dimeric E3 component are dissociated into monomers in the presence of 6 M GdnHCl; the latter enzyme forms higher-M(r) aggregates under these conditions.

Animals↗