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PubMed1985

Purification and Characterization of the Pea Chloroplast Pyruvate Dehydrogenase Complex : A Source of Acetyl-CoA and NADH for Fatty Acid Biosynthesis.

The pyruvate dehydrogenase complex has been purified 76-fold, to a specific activity of 0.6 mumoles per minute per milligram protein, beginning with isolated pea (Pisum sativum L. var Little Marvel) chloroplasts. Purification was accomplished by rate zonal sedimentation, polyethyleneglycol precipitation, and ethyl-agarose affinity chromatography. Characterization of the substrates as pyruvate, NAD(+), and coenzyme-A and the products as NADH, CO(2), and acetyl-CoA, in a 1:1:1 stoichiometry unequivocally established that activity was the result of the pyruvate dehydrogenase complex. Immunochemical analysis demonstrated significant differences in structure and organization between the chloroplast pyruvate dehydrogenase complex and the more thoroughly characterized mitochondrial complex. Chloroplast complex has a higher magnesium requirement and a more alkaline pH optimum than mitochondrial complex, and these properties are consistent with light-mediated regulation in vivo. The chloroplast pyruvate dehydrogenase complex is not, however, regulated by ATP-dependent inactivation. The properties and subcellular localization of the chloroplast pyruvate dehydrogenase complex are consistent with its role of providing acetyl-CoA and NADH for fatty acid synthesis.

Journal Article↗

PubMed2006

Regulatory roles of the N-terminal domain based on crystal structures of human pyruvate dehydrogenase kinase 2 containing physiological and synthetic ligands.

Pyruvate dehydrogenase kinase (PDHK) regulates the activity of the pyruvate dehydrogenase multienzyme complex. PDHK inhibition provides a route for therapeutic intervention in diabetes and cardiovascular disorders. We report crystal structures of human PDHK isozyme 2 complexed with physiological and synthetic ligands. Several of the PDHK2 structures disclosed have C-terminal cross arms that span a large trough region between the N-terminal regulatory (R) domains of the PDHK2 dimers. The structures containing bound ATP and ADP demonstrate variation in the conformation of the active site lid, residues 316-321, which enclose the nucleotide beta and gamma phosphates at the active site in the C-terminal catalytic domain. We have identified three novel ligand binding sites located in the R domain of PDHK2. Dichloroacetate (DCA) binds at the pyruvate binding site in the center of the R domain, which together with ADP, induces significant changes at the active site. Nov3r and AZ12 inhibitors bind at the lipoamide binding site that is located at one end of the R domain. Pfz3 (an allosteric inhibitor) binds in an extended site at the other end of the R domain. We conclude that the N-terminal domain of PDHK has a key regulatory function and propose that the different inhibitor classes act by discrete mechanisms. The structures we describe provide insights that can be used for structure-based design of PDHK inhibitors.

Adenosine Triphosphate↗

PubMed1983

Phospholipids and the regulation of pyruvate dehydrogenase from rat adipocyte mitochondria.

Aqueous dispersions of 4 out of 9 phospholipids added individually to the mitochondrial fraction from rat adipocytes altered the activity of pyruvate dehydrogenase in a dose-dependent manner from 1 to 300 microM. Phosphatidylserine increased and phosphatidylcholine, phosphatidylinositol and phosphatidylinositol-4-phosphate decreased enzyme activity. The stimulation of pyruvate dehydrogenase induced by phosphatidylserine may be reversed to below basal activity by phosphatidylinositol-4-phosphate and to basal activity by NaF, a pyruvate dehydrogenase phosphatase inhibitor. The inhibition of pyruvate dehydrogenase induced by phosphatidylinositol-4-phosphate may be restored to basal levels by the addition of calcium. These results suggest that phosphatidylserine activates pyruvate dehydrogenase activity through activation of the phosphatase, perhaps forming a phosphatidylserine-calcium complex. The inhibition by phosphatidylinositol-4-phosphate may be mediated by disruption of the enzyme complex. The phospholipids may play a physiological role in the regulation of pyruvate dehydrogenase activity.

Adipose Tissue↗

PubMed1994

Identification of the dihydrolipoamide acetyltransferase subunit of the human pyruvate dehydrogenase complex as an autoantigen in halothane hepatitis. Molecular mimicry of trifluoroacetyl-lysine by lipoic acid.

Trifluoroacetylated (CF3CO-) proteins, elicited upon exposure of animals or humans to halothane, were recognized by anti-CF3CO antibody, monospecific for the hapten derivative N6-trifluoroacetyl-L-lysine. Anti-CF3CO antibodies cross-reacted with the dihydrolipoamide acetyltransferase (E2 subunit) of pyruvate dehydrogenase, indicating that epitopes on the E2 subunit of pyruvate dehydrogenase molecularly mimic those on CF3CO-proteins. Lipoic acid, the prosthetic group of the E2 subunit of pyruvate dehydrogenase was essential in this process, in that only the lipoylated form of the recombinantly expressed inner lipoyl domain of the human E2 subunit of pyruvate dehydrogenase, but not the unlipolyated form, was recognized by anti-CF3CO antibody. Furthermore, based on a high degree of structural relatedness, both CF3CO-Lys and (6RS)-lipoic acid, as well as the lipoylated peptide ETDK(lipoyl)ATIG specifically inhibited the recognition by anti-CF3CO antibody of the E2 subunit of pyruvate dehydrogenase, of trifluoroacetylated rabbit serum albumin and of human liver CF3CO-proteins. In sera of patients with halothane hepatitis, autoantibodies with properties identical to those of anti-CF3CO antibody were identified which could not discriminate between CF3CO-proteins and the E2 subunit of pyruvate dehydrogenase. These data suggest that the E2 subunit pyruvate of dehydrogenase is an autoantigen in halothane hepatitis and that molecular mimicry of CF3CO-proteins by the E2 subunit of pyruvate dehydrogenase is due to the similar structures of CF3CO-Lys and lipoic acid.

Acetyltransferases↗

PubMedDate not supplied

[Pyruvate dehydrogenase from pigeon breast muscle. Chemical modification of the enzyme associated with conformation changes in the protein molecule].

The two-phase character of essential histidine residues modification of pyruvate dehydrogenase component of pyruvate dehydrogenase complex from pigeon breast muscle by diethylpyrocarbonate has been demonstrated. The relative amplitude of the fast phase increases with increasing the modificator concentration. The model of chemical modification of dimeric enzyme where the modification of the residue in one subunit leads to the change of reactivity of corresponding residue in the other subunit is used for the description of inactivation kinetics. The expression for the diminishing of enzyme activity in the course of chemical modification and the methods of kinetic parameters estimation have been proposed. The following values of kinetic parameters for the modification of pyruvate dehydrogenase component by diethylpyrocarbonate were obtained (pH 6.0; 20 degrees C): k1 = 6400 +/- 400 M-1 min-1 (the microscopic rate constant for the modification of histidine residue in the intact dimer), k2 = 890 +/- 200 M-1 min-1 (the rate constant for the modification of histidine residue in the intact subunit in the dimer which contains one modified subunit) and kt = 0.9 +/- 0.2 min-1 (the rate constant for conformational transition of the dimer induced by modification of histidine residue in one of the subunits in the dimeric molecule).

Animals↗

PubMed2000

Starvation increases the amount of pyruvate dehydrogenase kinase in several mammalian tissues.

Covalent modification of the pyruvate dehydrogenase complex provides an important regulatory mechanism for controlling the disposal of glucose and other compounds metabolized to pyruvate. Regulation of the complex by this mechanism is achieved in part by tissue-specific expression of the genes encoding isoenzymes of pyruvate dehydrogenase kinase (PDK). Starvation is known from our previous work to increase PDK activity of heart and skeletal muscle by increasing the amount of PDK isoenzyme 4 (PDK4) present in these tissues. This study demonstrates that increased expression of both PDK4 and PDK2 occurs in rat liver, kidney, and lactating mammary gland in response to starvation. PDK4 and PDK2 message levels were also increased by starvation in the two tissues examined (liver and kidney), suggesting enhancement of gene transcription. Changes in PDK2 message and protein were of similar magnitude, but changes in PDK4 message were greater than those in PDK4 protein, suggesting regulation at the level of translation. In contrast to these tissues, starvation had little or no effect on PDK2 and PDK4 protein in brain, white adipose tissue, and brown adipose tissue. Nevertheless, PDK4 message levels were significantly increased in brain and white adipose tissue by starvation. The findings of this study indicate that increased expression of PDK isoenzymes is an important mechanism for bringing about inactivation of the pyruvate dehydrogenase complex during starvation in many but not all tissues of the body. The absence of this mechanism preserves the capacity of neuronal tissue to utilize glucose for energy during starvation.

Animals↗

PubMed1992

Mutation of E1 alpha gene in a female patient with pyruvate dehydrogenase deficiency due to rapid degradation of E1 protein.

A mutation of an insertion of 4 bp in the gene for the alpha subunit of pyruvate dehydrogenase (E1 alpha) was found in a female with pyruvate dehydrogenase deficiency due to the rapid degradation of alpha and beta subunit proteins of pyruvate dehydrogenase. This mutation caused a frameshift that altered the amino acid sequence and created a premature stop codon. This 4-bp insertion has been found in an unrelated female patient with E1 alpha deficiency. It is rare that the same mutation is found in unrelated patients with this rare inborn error of metabolism. Furthermore, short deletions or duplications in the E1 alpha gene of patients with E1 alpha deficiency have been found only in exons 10 and 11. These exons may be hot spots for the mutations by the recombinational processes. This patient was heterozygous for the normal and a mutant allele. However, in most of the cultured skin fibroblasts from this patient, the mutant allele was expressed. These observations suggest that the X chromosome containing the normal allele was predominantly inactivated so that she developed lactic acidaemia and neurological abnormalities despite being heterozygous. The mutant alpha subunit protein failed to form a stable structure of pyruvate dehydrogenase, so that both alpha and beta subunit proteins were degraded rapidly.

Alleles↗

PubMed1993

Human pyruvate dehydrogenase complex as an autoantigen in primary biliary cirrhosis.

1. The sera of more than 90% of patients with primary biliary cirrhosis contain antimitochondrial antibodies which react with the E2 component of the pyruvate dehydrogenase complex, identified as the major autoantigen in primary biliary cirrhosis. All previous studies in this area have utilized protein derived from animal tissue or have used recombinant human pyruvate dehydrogenase complex E2 expressed in Escherichia coli. 2. We report the preparation and characterization of native pyruvate dehydrogenase complex and pyruvate dehydrogenase complex E2 from human heart tissue and its application in studies of immune reactivity with the sera of patients with primary biliary cirrhosis. 3. The immune reactivity of sera from patients with primary biliary cirrhosis versus the bovine and human E2/X components of pyruvate dehydrogenase complex was indistinguishable in both immunoblotting and the more sensitive e.l.i.s.a. 4. These findings suggest that the reactivity of sera from patients with primary biliary cirrhosis against the major autoantigen of the disease is a property of that antigen, independent of its human or bovine origin. Furthermore, this justifies the use of bovine pyruvate dehydrogenase complex in past and future work on primary biliary cirrhosis antibody reactivity.

Animals↗

PubMed2000

Import, processing, and assembly of the alpha- and beta-subunits of chloroplast pyruvate dehydrogenase.

Sequence comparisons were used to identify cDNAs potentially encoding the alpha- and beta-subunits of chloroplast pyruvate dehydrogenase. Coupled in-vitro transcription plus translation was used to synthesize radiolabeled pyruvate dehydrogenase alpha- and beta-subunit precursor proteins. When the precursors were incubated with intact pea (Pisum sativum L.) seedling chloroplasts in the presence of ATP, they were imported and proteolytically processed. In contrast, there was no import or processing of the precursors by isolated, intact pea seedling mitochondria. Monospecific antibodies to the recombinant pyruvate dehydrogenase alpha-subunit were additionally able to co-precipitate radiolabeled pyruvate dehydrogenase beta-subunit, indicating association between subunits after import and processing. Furthermore, size-exclusion chromatography was used to identify an alphabeta heterodimer that is an intermediate in the assembly of the native alpha2beta2 heterotetrameric enzyme.

Chloroplasts↗

PubMed1988

Estimation of pyruvate dehydrogenase (E1) activity in human skeletal muscle; three cases with E1 deficiency.

Pyruvate dehydrogenase (E1) catalyzes the rate-limiting step of the pyruvate dehydrogenase complex. Since E1 activity of human muscle tissue is low, a sensitive method is needed for diagnostic purposes. Measurement of 14CO2 production from [1-14C]pyruvate provides a specific and sensitive assay for measuring E1 activity. We use as artificial electron acceptor dichlorophenolindophenol (DCPIP) instead of the often applied ferricyanide. The method can be applied to small muscle samples obtained by needle or open biopsy. We prefer to use total homogenate because E1 activities in homogenate are higher than in the corresponding 600-g supernatant of skeletal muscle tissue. Control values in homogenate are higher or of the same order as those reported by others.

Adolescent↗

PubMed1991

Cryoelectron microscopy of mammalian pyruvate dehydrogenase complex.

Cryoelectron microscopy has been performed on frozen-hydrated pyruvate dehydrogenase complexes from bovine heart and kidney and on various subcomplexes consisting of the dihydrolipoyl transacetylase-based (E2) core and substoichiometric levels of the other two major components, pyruvate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3). The diameter of frozen-hydrated pyruvate dehydrogenase complex (PDC) is 50 nm, which is significantly larger than previously reported values. On the basis of micrographs of the subcomplexes, it is concluded that the E1 and E3 are attached to the E2-core complex by extended (4-6 nm maximally) flexible tethers. PDC constructed in this manner would probably collapse and appear smaller than its native size when dehydrated, as was the case in previous electron microscopy studies. The tether linking E1 to the core involves the hinge sequence located between the E1-binding and catalytic domains in the primary sequence of E2, whereas the tether linking E3 is probably derived from a similar hinge-type sequence in component X. Tilting of the E2-based cores and comparison with model structures confirmed that their overall shape is that of a pentagonal dodecahedron. The approximately 6 copies of protein X present in PDC do not appear to be clustered in one or two regions of the complex and are not likely to be symmetrically distributed.

Acetyltransferases↗

PubMed1982

Activation by exercise of human skeletal muscle pyruvate dehydrogenase in vivo.

1. The activity of pyruvate dehydrogenase in its active and inactive forms was measured in biopsy samples obtained from the vastus lateralis muscle of healthy subjects before and after exercise. 2. At rest, 40 +/- 4% (mean +/- SEM) of the enzyme was in the active form. 3. After progressive aerobic exercise to exhaustion (n = 5), 88 +/- 2.3% was in the active form. 4. After intermittent supramaximal short-term exercise (1 min exercise, 3 min rest) to exhaustion (n = 6), 60 +/- 2.2% was in the active form. 5. After isometric maximal exercise of 65 +/- 3.6 s duration (n = 3), only 39 +/- 1% of the enzyme was in the active form. 6. Muscle glycogen depletion was greatest with intermittent exercise and least with isometric maximal exercise; in contrast, the increase in muscle lactate was least with progressive exercise (1.3 to 9.4 mumol/g), intermediate in intermittent maximal exercise (1.2 to 13.1 mumol/g) and greatest after isometric exercise (1.8 to 17.6 mumol/g). There were no significant differences between the three studies in the changes in lactate/pyruvate ratios. 7. In three subjects who exercised with one leg, activation of the enzyme was twice as great in the exercise as in the inactive leg. 8. The ratio of active to total enzyme in biopsies of resting muscle was greater in four well-trained athletes than in four untrained control subjects (70% compared with 41% respectively). 9. The activation of pyruvate dehydrogenase appears to play an important part in regulating the use of glycogen and glucose during exercise in man.

Adult↗

PubMed1985

Changes in the activity of 'active' pyruvate dehydrogenase complex in the newborn of normal and diabetic rats.

At birth, hepatic 'active' and 'dichloracetate-activated' pyruvate dehydrogenase complex activities in the newborn of normal, mildly diabetic, and severely diabetic rats were similar. The 'active' and 'dichloracetate-activated' pyruvate dehydrogenase complex activities increased significantly during the first 2 and 6 postnatal h, respectively in the three groups of neonates (p less than 0.05). The greatest increase in both 'active' and 'dichloroacetate-activated' pyruvate dehydrogenase complex activity was observed in the neonates of mildly diabetic rats. Administration of glucose or insulin at birth to the newborn of normal rats caused a significant increase in the percentage of 'active' pyruvate dehydrogenase complex activity within 1 h (p less than 0.01). Similar treatment caused no significant increases in the newborn of severely diabetic rats. The transient increases in 'active' pyruvate dehydrogenase complex activity in the neonates of normal and diabetic rats were consistent with rapid disappearance of blood lactate during the first hours of postnatal life.

Animals↗

PubMed1988

Structure function studies on the lipoate-acetyltransferase--component-X-core assembly of the ox heart pyruvate dehydrogenase complex.

Component X, the recently recognised subunit of mammalian pyruvate dehydrogenase complex, was shown by immune blotting to be present in all of nine tissues dissected from rat. This finding indicated that component X was not an isoenzyme of the lipoate acetyltransferase (E2) associated with one or a limited number of tissues. Native pyruvate dehydrogenase complex was shown to bind IgG raised to isolated component X, indicating that there were at least some regions of the X subunit exposed at the periphery of the complex. Lipoyl groups of ox heart pyruvate dehydrogenase complex were specifically cross-linked by reaction with phenylene-o-bismaleimide in the presence of pyruvate and the subunits contributing to the products of cross-linking were identified by immune blotting. Species with very high Mr containing both E2 and component X, were formed in high yield, as well as apparent E2/E2 and E2/X dimers and trimers and an X/X dimer. These results showed that acetylated lipoyl groups of different E2 and X subunits were able to interact in all possible combinations. The types of cross-linked E2 products formed suggested that two thiols, reactible with phenylene-o-bismaleimide, were rapidly generated in the presence of pyruvate. The results were most easily explained by the presence of two acetylatable lipoyl groups on each E2 polypeptide.

Acetyltransferases↗

PubMed1995

An amino acid substitution in the pyruvate dehydrogenase E1 alpha gene, affecting mitochondrial import of the precursor protein.

A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1 alpha deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1 alpha immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1 alpha targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1 alpha sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease.

Amino Acids↗

PubMed1982

Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney.

Pyruvate dehydrogenase phosphatase was purified to apparent homogeneity from bovine heart and kidney mitochondria. The phosphatase has a sedimentation coefficient (S20,w) of about 7.4 S and a molecular weight (Mr) of about 150 000 as determined by sedimentation equilibrium and by gel-permeation chromatography. The phosphatase consists of two subunits with molecular weights of about 97 000 and 50 000 as estimated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Phosphatase activity resides in the Mr 50 000 subunit, which is sensitive to proteolysis. The phosphatase contains approximately 1 mol of flavin adenine dinucleotide (FAD) per mol of protein of Mr 150 000. FAD is apparently associated with the Mr 97 000 subunit. The function of this subunit remains to be established. The phosphatase binds 1 mol of Ca2+ per mol of enzyme of Mr 150 000 at pH 7.0, with a dissociation constant (Kd) of about 35 microM as determined by flow dialysis. Use of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) at pH 7.6 in conjunction with flow dialysis gave a Kd value for Ca2+ of about 8 microM. In the presence of both the phosphatase and the dihydrolipoyl transacetylase (E2) core of the pyruvate dehydrogenase complex, two equivalent and apparently non-interacting CA2+-binding sites were detected per unit of Mr 150 000, with a Kd value of about 24 microM in the absence and about 5 microM in the presence of EGTA. In the presence of 0.2 M KCl, which inhibits phosphatase activity about 95%, the phosphatase exhibited only one Ca2+-binding site, even in the presence of E2. The phosphatase apparently possesses an "intrinsic" Ca2+-binding site, and a second Ca2+-binding site is produced in the presence of E2. The second site is apparently altered by increasing the ionic strength. It is proposed that the second site may be at the interface between the phosphatase and E2, with Ca2+ acting as a bridging ligand for specific attachment of the phosphatase to E2.

Animals↗

PubMed1985

[Pyruvate dehydrogenase deficiency in a child with persistent lactic acidosis].

Evidence is presented for defective pyruvate dehydrogenase (EC 4.1.1.1) in leukocytes and muscle tissue from a 10-year old child with persistent lactic acidosis, suffering from myasthenia and growth retardation. The defect is expressed in vitro by a depressed stimulation of pyruvate dehydrogenase catalytic activity by exogenous phosphoprotein phosphatase, and in vivo by a lack of response to muscle work, in comparison with healthy controls. Pyruvate dehydrogenase activity is in the normal range when measured without addition of phosphoprotein phosphatase in cells obtained from the resting patient. The defect reported here represents a new, hitherto undescribed form of a pyruvate dehydrogenase deficiency. The insufficient catalytic activity explains the observed accumulation of pyruvate, lactate, oxaloacetate and alanine and the decrease of citrate concentration in the blood of this patient. Electron microscope studies of the muscle tissue show an enhanced number of enlarged mitochondria with bizarre shapes and high densities of cristae.

Acidosis↗

PubMed1980

The pyruvate dehydrogenase complex of Pseudomonas aeruginosa PAO Purification, properties and characterization of mutants.

The pyruvate dehydrogenase complex of Pseudomonas aeruginosa PAO was purified by affinity chromatography on ethanol-Sepharose 2B followed by sucrose density gradient centrifugation. The overall purification was 130-fold based on enzyme activity. The purified complex contained three major and one minor polypeptide components when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These were identified by heat treatment, limited proteolysis and peptide mapping as pyruvate dehydrogenase (El; Mr 92500), acetyltransferases (E2; major component, Mr 76000, and minor component, Mr 77800) and lipoamide dehydrogenase (E3; Mr 58000). The purified complex had a sedimentation coefficient of 48S and the specific activity for the overall reaction of the complex was 6.5 micromol substrate transformed (mg protein)-1 min-1 at the optimum pH (7.8) and 25 degrees C. The lesions in four ace mutants lacking overall pyruvate dehydrogenase complex activity were identified after partial purification of the corresponding cell-free extracts. Three strains, designated ace A mutants, lacked pyruvate dehydrogenase activity (E1 component) and one strain, and ace B mutant, lacked the activity of the acetyltransferase (E2 component).

Antibodies↗