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PubMed1986

Improved purification of the pyruvate dehydrogenase complex from Ascaris suum body wall muscle and characterization of PDHa kinase activity.

An improved purification scheme for the isolation of the Ascaris suum pyruvate dehydrogenase complex directly from body wall muscle has been developed which yields a fully activated pyruvate dehydrogenase complex with substantial PDHa kinase activity. The apparent Km for coenzyme A (CoA) is much lower than previously reported and can only be accurately measured in the presence of a CoA-regenerating system. The alpha-pyruvate dehydrogenase subunit of the ascarid complex is unique and its migration on sodium dodecylsulfate polyacrylamide gels is altered after phosphorylation. PDHa kinase activity is inhibited by ADP, thiamine pyrophosphate, and physiological levels of pyruvate and propionate. In contrast, PDHa kinase activity is stimulated by elevated NADH/NAD+ and acetyl CoA/CoA ratios, although it appears that the NADH/NAD+ ratios required for half-maximal stimulation are more than an order of magnitude greater than those reported for mammalian pyruvate dehydrogenase complexes.

Acetyl Coenzyme A↗

PubMed1999

A model for the spatial location of pyruvate dehydrogenase phosphatase in mammalian pyruvate dehydrogenase complex.

Recent experimental findings on the structural--functional features of pyruvate dehydrogenase phosphatase (PDP) isolated from various sources are compared. Two alternative mechanisms (a and b) of dephosphorylation of the E1 component in the pyruvate dehydrogenase complex (PDC) are discussed: a) the reaction occurs as a result of stochastic collisions of PDP and PDC, and the generation of an enzyme--substrate complex (PDP--E1--PDC) and dephosphorylation of the E1 component occur independently at different PDP binding sites on the PDC core; b) the dephosphorylation is performed simultaneously by a certain number of PDP molecules symmetrically bound on the PDC core. The second mechanism is suggested by the self-assembly theory of multicomponent enzyme systems and can be proved by kinetic experiments. Based on self-assembly principles and data on feasible binding sites of peripheral components of the PDC, the stoichiometry and mutual location of PDP, pyruvate dehydrogenase kinase, and the E1 component on the core of mammalian PDC are postulated to provide optimal functioning of the PDC. Structural mechanisms of stimulation of PDP activity by Ca2+ and polyamines are also discussed.

Animals↗

PubMed1981

An NADH-linked spectrophotometric assay for pyruvate dehydrogenase complex in crude tissue homogenates.

A spectrophotometric assay for the pyruvate dehydrogenase complex has been developed, which measures NADH production by coupling it to the reduction of a tetrazolium dye via an intermediate electron carrier. The procedure uses only commercial reagents. Dependence on pyruvate, coenzyme A, and NAD is complete, and activities are comparable to those obtained by other procedures with both purified (rat kidney mitochondria) and crude (mouse brain homogenate) preparations. These dye-coupled procedures are potentially applicable to assays of other NADH-producing dehydrogenases in crude preparations.

Animals↗

PubMed1994

Enzyme inhibitory autoantibodies to pyruvate dehydrogenase complex in primary biliary cirrhosis differ for mammalian, yeast and bacterial enzymes: implications for molecular mimicry.

Primary biliary cirrhosis is a chronic autoimmune disease in which serum autoantibodies against the mitochondrial 2-oxo acid dehydrogenase enzyme complexes (M2 antibodies) are regularly present. Molecular mimicry of host proteins by bacterial counterparts is a suggested explanation for the origin of these autoantibodies. We tested this hypothesis by measuring the functional reactivity of serum autoantibodies by means of an enzyme inhibition assay against pyruvate dehydrogenase complex from different sources: mammalian, Saccharomyces cerevisiae and Escherichia coli. The 10 primary biliary cirrhosis sera all reacted on immunofluorescence study for M2 antibodies and on immunoblotting with the pyruvate dehydrogenase complex E2 subunit from each of the three enzymes, but there were strikingly different inhibitory capacities. The primary biliary cirrhosis sera were highly inhibitory for mammalian pyruvate dehydrogenase complex (10 of 10 inhibitory; mean level of inhibition, 99%), moderately inhibitory for yeast pyruvate dehydrogenase complex (10 of 10 inhibitory; mean level, 70%) and weakly inhibitory for Escherichia coli pyruvate dehydrogenase complex (4 of 10 inhibitory; mean level, 26%). Thus, with a functional assay that depends on epitope recognition of primary biliary cirrhosis sera, cross-reactivity between mammalian and bacterial pyruvate dehydrogenase complex enzymes is low and molecular mimicry, at least at the B-lymphocyte level, is not supported.

Animals↗

PubMed1999

Exceptional characteristics of heterotetrameric (alpha 2 beta 2) E1p of the pyruvate dehydrogenase complex from Zymomonas mobilis: expression from an own promoter and a lipoyl domain in E1 beta.

In the pyruvate dehydrogenase complex (PDHC) of Zymomonas mobilis the beta subunit of the pyruvate dehydrogenase (E1p) as well as the acetyltransferase (E2p) contain an N-terminal lipoyl domain. Both lipoyl domains were acetylated in vitro using 2-14C-pyruvate as a substrate, demonstrating that both lipoyl domains can accept acetyl groups from the E1 component. As previously shown the structural genes (pdhA alpha beta, pdhB, lpd) encoding the pyruvate dehydrogenase complex of Z. mobilis are located in two distinct gene clusters, pdhA alpha beta and pdhB-orf2-lpd (U. Neveling et al. (1998) J. Bacteriol. 180, 1540-1548). Analysis of pdh gene expression using lacZ fusions revealed that the DNA fragments upstream of pdhA alpha, pdhB and lpd each have promoter activities. These pdh promoter activities were 7-30-fold higher in Z. mobilis than in Escherichia coli.

Kinetics↗

PubMed1998

Detection of a homozygous four base pair deletion in the protein X gene in a case of pyruvate dehydrogenase complex deficiency.

While the presence of a lipoyl-containing protein (protein X) separate from lipoyl transacetylase in the pyruvate dehydrogenase complex (PDC) has been known for some time, until recently only the cDNA for the yeast enzyme has been cloned. We have cloned, sequenced and characterized the cDNA encoding the human protein X and localized the protein X gene to chromosome 11p13. We also report here a new case of protein X deficiency identified immunologically, with decreased activity of PDC and without mutations in the E1alpha subunit or E1beta subunit. We report that the cDNA and gene of this patient for protein X has a homozygous 4 bp deletion, specifically in the putative mitochondrial targeting signal sequence which results in a premature stop codon. This is the first documented case of a molecular defect in pyruvate dehydrogenase protein X.

Amino Acid Sequence↗

PubMed1995

Cloning and characterization of a dihydrolipoamide acetyltransferase (E2) subunit of the pyruvate dehydrogenase complex from Arabidopsis thaliana.

A cDNA encoding a dihydrolipoamide acetyltransferase (E2) subunit of the pyruvate dehydrogenase complex has been isolated from Arabidopsis thaliana. A cell culture cDNA expression library was screened with a monoclonal antibody (JIM 63) raised against nuclear matrix proteins, and four clones were isolated. One of these was 2175 base pairs in length, and it contained an open reading frame with an amino acid sequence and domain structure with strong similarity to the E2s of other eukaryotic and prokaryotic organisms. The organization and number of functional domains within the Arabidopsis protein are identical to those of the human E2, although the amino acid sequences within these domains are equally similar to those of the yeast and human proteins. The predicted amino acid sequence reveals the presence of a putative amino-terminal leader sequence with characteristics similar to those of other proteins, which are targeted to the plant mitochondrial matrix. The cross-reactivities of plant mitochondrial matrix proteins with JIM 63 and antibodies raised against the E2 and protein X components of eukaryotic pyruvate dehydrogenase complexes are consistent with the clone encoding a mitochondrial form of E2 and not the smaller protein X. The E2 mRNA of 2.2 kilobases was expressed in a range of Arabidopsis and Brassica napus tissues.

Acetyltransferases↗

PubMed1995

Recombinant expression and evaluation of the lipoyl domains of the dihydrolipoyl acetyltransferase component of the human pyruvate dehydrogenase complex.

The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) associate to form a large inner core with a protruding structure composed of three globular domains connected by mobile linker regions. This exterior region of E2 includes two lipoyl domains which engage not only in the intermediate reactions of the complex but also have integral roles in the kinase-phosphatase regulatory interconversion of the pyruvate dehydrogenase (E1) component. To facilitate understanding of these roles, lipoyl domain constructs of the E2 component of human PDC were expressed as glutathione S-transferase (GST)-linked fusion proteins from plasmid inserts prepared by polymerase chain reaction procedures. The NH2-terminal lipoyl domain, E2L1, and the interior lipoyl domain, E2L2, are connected by a 30-amino-acid hinge region, H1. Constructs designed and expressed were E2L1(1-98), E2L1.H1(1-128), E2L2(120-233), E2H1.L2(98-233), and E2L1.H1.L2(1-233), where numbers in parentheses give the amino acid sequence for the portions of the E2 component incorporated into a construct. The domains were expressed in Escherichia coli with and without lipoate supplementation. GST constructs were purified to homogeneity by affinity chromatography and selectively released by thrombin treatment. Sequencing of insert DNAs and NH2-terminal sequencing confirmed that domains were produced as designed. Measurement of masses by electrospray mass spectrometry indicated that constructs with lipoylated, nonlipoylated, and octanoylated forms were produced when expression was with E. coli grown without lipoate supplementation and that fully lipoylated forms were produced upon lipoate supplementation. The lipoylation status was confirmed, following delipoylation with Enterococcus faecalis lipoamidase, by the expected decrease in mass and by the observation in native gel electrophoresis of a shift to a slower mobility (possibly less compact) form. Constructs were used in E1-catalyzed reductive-acetylation reaction in proportion to their degree of lipoylation and were effective substrates in a NADH-dependent dihydrolipoyl dehydrogenase reduction reaction. Thus, we have produced lipoyl domain constructs that can be employed in sorting the specific roles of E2L1 and E2L2 in facilitating catalytic and regulatory processes.

Acetylation↗

PubMed1991

Inhibitory autoantibody to a conformational epitope of the pyruvate dehydrogenase complex, the major autoantigen in primary biliary cirrhosis.

The mitochondrial autoantibodies present in primary biliary cirrhosis (PBC) react with the 2-oxoacid dehydrogenase enzymes that include the pyruvate dehydrogenase complex (PDC). All epitopes so far demonstrable, including the inner lipoyl domain of PDC-E2, have been revealed by immunoblotting. To identify other epitopes, advantage was taken of the capacity of PBC sera to inhibit in vitro the catalytic function of the PDC enzyme. PBC sera were analyzed by affinity chromatography, using columns containing either recombinant PDC-E2 or intact PDC. Fractions that bound to the column (B) and nonbinding effluent fractions (NB) were tested by immunoblotting and ELISA and for their capacity to inhibit enzyme function. After separation on the PDC-E2 column the B fractions were reactive with PDC-E2 and intact PDC, whereas the NB fractions did not react by immunoblotting or ELISA with PDC-E2 but did react strongly by ELISA with PDC and did strongly inhibit the enzyme function. After separation of sera on the PDC column, the B fractions reacted more strongly with PDC than PDC-E2 by ELISA and strongly inhibited the enzyme function, whereas the NB fractions were nonreactive. Thus we describe a hitherto undetected population of autoantibodies in PBC sera that react only with intact PDC but not with the recombinant PDC-E2 subunit that contains the lipoyl epitope, are demonstrable by ELISA but not by immunoblotting, and notably, inhibit enzyme function. These nonblotting inhibitory autoantibodies in PBC are presumed to react with an exclusively conformational determinant perhaps presented by the tertiary structure of the entire enzyme complex.

Autoantibodies↗

PubMed1986

Effect of sepsis on activity of pyruvate dehydrogenase complex in skeletal muscle and liver.

The effect of chronic sepsis on the concentration of active pyruvate dehydrogenase complex has been investigated in liver and skeletal muscle of normal, sterile inflammatory, and chronic septic (small and large abscess) animals. Hyperdynamic sepsis was induced by the intraperitoneal introduction of a rat fecal-agar pellet of known size and bacterial composition (Escherichia coli + Bacteroides fragilis). Total pyruvate dehydrogenase complex activity was not altered in either liver or skeletal muscle in any of the conditions studied. In hepatic tissue, sterile inflammation increased the proportion of active complex 2.5-fold compared with control. The same increase in the concentration of active complex was observed in animals with a small abscess. When the abscess size was increased (large abscess), the concentration of active complex was decreased relative to sterile inflammatory or small abscess septic animals. In contrast to liver, sterile inflammation did not alter the proportion of active complex in skeletal muscle. Sepsis (either small or large septic abscess) resulted in threefold decrease in the concentration of active complex relative to control or sterile inflammatory animals. Changes in the concentration of active complex did not appear to be dependent on the ATP/ADP concentration ratio or tissue pyruvate levels but were consistent with changes in the acetyl-coenzyme A-to-coenzyme A concentration ratio. The mechanism responsible for altered concentration of active complex may be mediated through changes in the activity of the pyruvate dehydrogenase kinase, secondary to alterations in the effector concentration ratios.

Abscess↗

PubMed1994

Biochemical and molecular characterization of the Alcaligenes eutrophus pyruvate dehydrogenase complex and identification of a new type of dihydrolipoamide dehydrogenase.

Sequence analysis of a 6.3-kbp genomic EcoRI-fragment of Alcaligenes eutrophus, which was recently identified by using a dihydrolipoamide dehydrogenase-specific DNA probe (A. Pries, S. Hein, and A. Steinbüchel, FEMS Microbiol. Lett. 97:227-234, 1992), and of an adjacent 1.0-kbp EcoRI fragment revealed the structural genes of the A. eutrophus pyruvate dehydrogenase complex, pdhA (2,685 bp), pdhB (1,659 bp), and pdhL (1,782 bp), encoding the pyruvate dehydrogenase (E1), the dihydrolipoamide acetyltransferase (E2), and the dihydrolipoamide dehydrogenase (E3) components, respectively. Together with a 675-bp open reading frame (ORF3), the function of which remained unknown, these genes occur colinearly in one gene cluster in the order pdhA, pdhB, ORF3, and pdhL. The A. eutrophus pdhA, pdhB, and pdhL gene products exhibited significant homologies to the E1, E2, and E3 components, respectively, of the pyruvate dehydrogenase complexes of Escherichia coli and other organisms. Heterologous expression of pdhA, pdhB, and pdhL in E. coli K38(pGP1-2) and in the aceEF deletion mutant E. coli YYC202 was demonstrated by the occurrence of radiolabeled proteins in electropherograms, by spectrometric detection of enzyme activities, and by phenotypic complementation, respectively. A three-step procedure using chromatography on DEAE-Sephacel, chromatography on the triazine dye affinity medium Procion Blue H-ERD, and heat precipitation purified the E3 component of the A. eutrophus pyruvate dehydrogenase complex from the recombinant E. coli K38(pGP1-2, pT7-4SH7.3) 60-fold, recovering 41.5% of dihydrolipoamide dehydrogenase activity. Microsequencing of the purified E3 component revealed an amino acid sequence which corresponded to the N-terminal amino acid sequence deduced from the nucleotide sequence of pdhL. The N-terminal region of PdhL comprising amino acids 1 to 112 was distinguished from all other known dihydrolipoamide dehydrogenases. It resembled the N terminus of dihydrolipoamide acyltransferases, and it contained one single lipoyl domain which was separated by an adjacent hinge region from the C-terminal region of the protein that exhibited high homology to classical dihydrolipoamide dehydrogenases.

Alcaligenes↗

PubMed1995

Changes in the structure of pyruvate dehydrogenase complex induced by mono- and divalent ions.

The activity of pyruvate dehydrogenase complex purified from pig kidney cortex was affected by various mono- and di-valent ions and changes in ionic strength. The fluorescence emission spectrum of PDC exposed to 0.04 M ionic strength and excited at 280 nm exhibited a maximum at 334 nm; the fluorescence intensity of PDC appeared to depend upon the ionic strength and the K+ and Na+ content of the incubation buffer. Alteration of ionic strength to which the enzyme complex was exposed produced a change in the absorption of the complex at 230 nm. The presence of HPO4(2-) ions prevented changes in the UV absorption spectrum of PDC induced by the variation in ionic strength. The K+ and Na+ ions alone had no effect on the UV spectrum of PDC. Upon increasing the ionic strength to which the enzyme complex was exposed, dramatic changes in the circular dichroism (CD) pattern were observed. At 0.04 M ionic strength PDC exhibited a CD spectrum with minima at 216, 218 and 222 nm and a cross-over point at 215 nm. At 0.15 M ionic strength the CD spectrum of PDC exhibited minima at 223, 226, 228 nm and a cross-over point at 221 nm. The presence of HPO4(2-) ions prevented alterations in the CD spectrum of PDC induced by variations in ionic strength. The K+ and Na+ ions had no effect on the CD spectrum of PDC.

Animals↗

PubMed1982

Sepharose-insolubilization of the dihydrolipoyl transacetylase core component of the pyruvate dehydrogenase complex: preparation and characterization.

The dihydrolipoyl transacetylase core components of the bovine kidney and heart pyruvate dehydrogenase complexes were covalently attached through the lipoyl moiety to Sepharose by the thiol-crosslinking reagent, N,N'-p-phenylenedimaleimide. In one approach, the N,N-p-phenylenedimaleimide was allowed to react with glutathione which was in turn linked by its N-terminal to Sepharose CL-6B. In addition, we found that N,N-p-phenylenedimaleimide would react directly with Sepharose CL-6B (at undetermined sites) and could be used as the sole bridge in forming a stable linkage of the transacetylase core to Sepharose. With the latter approach the extent of multiple-linkage of the 60-subunit core could more easily be controlled. This should be a generally useful approach for linking proteins with reactive surface thiol residues. Insolubilization of the core of the pyruvate dehydrogenase complex by these methods did not appear to significantly alter the binding of other protein components of the complex, but the catalytic activities of the complex requiring the lipoyl moiety were appreciably altered. Procedures for coupling the transacetylase core to various derivatives of phenylenedimaleimide-Sepharose and techniques described for studying the protein products should be useful in preparation of specialized matrices for both protein purification and the study of protein-protein interactions.

Acetyltransferases↗

PubMed1990

Isolation, characterization and chromosomal localization of cDNA clones for the E1 beta subunit of the pyruvate dehydrogenase complex.

A full-length cDNA clone for the E1 beta subunit of the human pyruvate dehydrogenase (PyrDH) complex was isolated from a human skin fibroblast cDNA library. When sequenced, it showed differences from the nucleotide sequence already published [Koike, K., Ohta, S., Urata, Y., Kagawa, Y. & Koike, M. (1988) Proc. Natl Acad. Sci. USA 85, 41-45], such that 19 amino acids were different in the translated open reading frame. Northern blotting of human fibroblast cell lines revealed a major mRNA species of 1.6 kb and a weaker band of 5.5 kb. In a series of nine PyrDH-complex-deficient cell lines from patients with this deficiency, no patients had severely reduced amounts of mRNA, but there was one patient cell line with an increased amount of abnormal-size mRNA. Chromosome localization carried out with DNA blots from man-mouse hybrid cell lines indicated that the E1 beta subunit of pyruvate dehydrogenase is located on chromosome 3. A motif AXGXXXXGL(R/K)X15(D/E)Q was found in common with a variety of other oxo-acid oxidoreductases, but its function is not known.

Amino Acid Sequence↗

PubMed1995

Application of anti-E1 monoclonal antibodies to the study of the pyruvate dehydrogenase complex.

It has been shown that monoclonal antibody (mAb) F7F10 raised against pyruvate dehydrogenase component (E1) of pigeon breast muscle pyruvate dehydrogenase complex (PDC) has no influence on the E1 activity, measured in the system with artificial oxidants. However it inhibited the full NAD+ and coenzyme A dependent activity of PDC. The competition of the F7F10 antibody with the E2 component of PDC for the binding with E1 was revealed by immunoenzymatic and kinetic analysis. It is suggested that F7F10 mAb interacts with an antigenic determinant, located in the immediate vicinity of or overlapping with the E1 region, responsible for the interaction with the E2 component of PDC.

Animals↗

PubMed2006

Assay of the pyruvate dehydrogenase complex by coupling with recombinant chicken liver arylamine N-acetyltransferase.

The activity of the pyruvate dehydrogenase complex has long been determined in some laboratories by coupling the production of acetyl-coenzyme A (acetyl-CoA) to the acetylation of 4-aminoazobenzene-4'-sulfonic acid by arylamine N-acetyltransferase. The assay has some advantages, but its use has been limited by the need for large amounts of arylamine N-acetyltransferase. Here we report production of recombinant chicken liver arylamine N-acetyltransferase and optimization of its use in miniaturized assays for the pyruvate dehydrogenase complex and its kinase.

Amino Acid Sequence↗

PubMed1997

Expression and characterisation of the homodimeric E1 component of the Azotobacter vinelandii pyruvate dehydrogenase complex.

We have cloned and sequenced the gene encoding the homodimeric pyruvate dehydrogenase component (E1p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and expressed and purified the E1p component in Escherichia coli. Cloned E1p can be used to fully reconstitute complex activity. The enzyme was stable in high ionic strength buffers, but was irreversibly inactivated when incubated at high pH, which presumably was caused by its inability to redimerize correctly. This explains the previously found low stability of the wild-type E1p component after resolution from the complex at high pH. Cloned E1p showed a kinetic behaviour exactly like the wild-type complex-bound enzyme with respect to its substrate (pyruvate), its allosteric properties, and its effectors. These experiments show that acetyl coenzyme A acts as a feedback inhibitor by binding to the E1p component. Limited proteolysis experiments showed that the N-terminal region of E1p was easily removed. The resulting protein fragment was still active with artificial electron acceptors but had lost its ability to bind to the core component (E2p) and thus reconstitute complex activity. E1p was protected against proteolysis by E2p. The allosteric effector pyruvate changed E1p into a conformation that is more resistant to proteolysis.

Amino Acid Sequence↗

PubMed1980

Isolation and properties of pyruvate dehydrogenase complex mutants of Pseudomonas aeruginosa PAO.

Four independent ace mutants of Pseudomonas aeruginosa PAO lacking the activity of the pyruvate dehydrogenase complex have been isolated. They resembled ace mutants of Escherichia coli and Salmonella typhimurium in requiring acetate as an essential supplement for aerobic growth on glucose, succinate or lactate and in their ability to utilize acetate as sole carbon and energy source. Assays for the individual components of the pyruvate dehydrogenase complex indicated that they lacked the pyruvate dehydrogenase component (El) or the pyruvate dehydrogenase and dihydrolipoamide acetyltransferase components (E1 and E2) but not the lipoamide dehydrogenase component (E3). Genetic studies with plasmid R68.45-mediated conjugation and phage F116L-mediated transduction indicated that the ace mutations are located at approximately 15 min in the P. aeruginosa PAO linkage map.

Acetates↗