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PubMedDate not supplied

[Effect of pyruvate dehydrogenase coenzymes and mitochondrial proteins on the accumulation of [35S]lipoic acid].

Coenzymes introduced in the ratio, peculiar for pyruvate dehydrogenase complex into the medium containing fresh-isolated mitochondria and oxidation substrate--pyruvate increase accumulation of [35S] lipoate by these organelles. This process is highly stimulated by introducing either the only CoA or a coenzyme mixture (CoA, thiamine pyrophosphate, FAD, NAD). Addition of phosphate-extracted components of mitochondria and their protein fraction with coenzymes in the ratio indicated above provides maximum accumulation of [35S] lipoate by liver mitochondria. An equimolar mixture of coenzymes as well as protein components evoke no reliable variations in [35S] lipoate accumulation by albino rat liver mitochondria, while addition of the only thiamine pyrophosphate decreases this accumulation. Reconstruction of multienzyme complexes of coenzymes and apoenzymes on mitochondrion membranes accounts for the results obtained.

Animals↗
PubMed1975

X-ray small-angle studies of the pyruvate dehydrogenase core complex from Escherichia coli K-12. II. Subunit structure of the core complex.

The interpretation of X-ray small-angle data of the pyruvate dehydrogenase core complex from E. coli K-12 reveals the fine structure of the complex. Specific inner surface (7.07-10(-2 A-1), inner surface (7.60 - 10(5) A2), MEAN TRANSVERSAL LENGTH (56.6 A), coherence length (123.5 A), structural factor (1.1), and coherence area (3.27 - 10(4) A2) have been determined as further structural parameters characterizing the colloidal distribution of matter. Fouier transformations of scattered intensity and of structural amplitude have been carried out and show the existence of slightly disturbed spherical symmetry of the complex built up from subunits. The mean diameter of the three different subunit components of about 78 A was determined from the correlation function or from the distance distribution. The number of subunits in the complex was ascertained to be 40. The radial excess electron density distribution shows the arrangement of the core complex from a "core" (formed by the transacetylase components) with a small hole inside and a "shell" (formed by the pyruvate dehydrogenase and dihydrolipoamide dehydrogenase components). Although not representing a unique solution, a lot of model calculations indicate how the complex is arranged from subunits. At each edge of a cubic centre, the edge formed by two chains of transacetylase, two chains of pyruvate dehydrogenase and two chains of dihydrolipoamide dehydrogenase components are arranged according to the best fit. Far-reaching conformity between experimental results and model was established.

Chemical Phenomena↗
PubMed1980

[Steady-state kinetics of the bovine adrenal pyruvate dehydrogenase complex-catalyzed reactions].

The hyperbolic dependence of the initial rate of the adrenal pyruvate dehydrogenase complex-catalyzed reactions on pyruvate, CoA and NAD concentrations was established. The Lineweaver--Burk plots of v0 against one substrate concentration at constant unsaturating concentrations of other substrates represent families of parallel lines. The Km values for pyruvate, CoA and NAD are 0,017, 0.005 and 0.030 mM, respectively. CoA-SAc was shown to compete with CoA (Ki=0.067 mM) whereas NADH--with NAD (Ki=0.023 mM). Both CoA-SAc and NADH are uncompetitive inhibitors with respect to pyruvate. The inhibition of CoA-SAc against NAD is of a mixed type, while that of NAD against CoA is non-competitive. The results obtained are in agreement with the kinetic model of a three-site "ping-pong" mechanism.

Adrenal Glands↗
PubMed1998

Systematic study of the six cysteines of the E1 subunit of the pyruvate dehydrogenase multienzyme complex from Escherichia coli: none is essential for activity.

Variants of the Escherichia coli 1-lip pyruvate dehydrogenase multienzyme complex (1-lip PDHc) with the C259N and C259S substitutions in the putative thiamin diphosphate-(ThDP-) binding motif of the pyruvate dehydrogenase component (E1, EC 1.2.4.1) were characterized. Single substitutions were made at the five remaining cysteines of the E1 component, creating the C120A, C575A, C610A, C654A, and C770S variants to test the hypothesis that the activity loss that accompanies exposure of the enzyme to fluoropyruvate, bromopyruvate, and 2-oxo-3-butynoic acid is the result of the modification of approximately one cysteine residue per E1 monomer. Surprisingly, all single cysteine E1 variants could be reconstituted with E2-E3 subcomplex and showed PDHc activity ranging from 74% to 96% that of the parental enzyme. The specific activities of C259N and C259S variants of 1-lip PDHc were 58% and 27% relative to that of the parental 1-lip PDHc. All five single cysteine E1 variants, along with the C259N and C259S variants of 1-lip PDHc, could also (1) be inactivated with fluoropyruvate and 2-oxo-3-butynoic acid, (2) were subject to inactivation by the monoclonal antibody 18A9 reported from one of our laboratories, and (3) were subject to regulation by pyruvate and acetyl-CoA. It was therefore concluded that none of the six cysteine residues is essential for the activity of the E1 component or of the complex. When tested with the putative transition-state analogue, thiamin 2-thiothiazolone diphosphate, all but the C259S and C259N variants were very potently inhibited, the stoichiometry for parental E1 being about 1.6 mol of inhibitor/mol of E1 subunit. The C259S and C259N E1 variants required at least 25-fold greater inhibitor concentration to achieve the same level of inhibition. C259 is located in the putative thiamin diphosphate-binding motif of the enzyme [more exactly, it is adjacent to a ligand to the Mg(II) ion]. It is therefore concluded that thiamin 2-thiothiazolone diphosphate is not a transition-state analogue; rather, it is a potent inhibitor of the complex because of a specific interaction with the C259 residue.

Acetyl Coenzyme A↗
PubMed1990

Cloning and sequence analysis of the genes encoding the alpha and beta subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

A 4175-bp EcoRI fragment of DNA that encodes the alpha and beta chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the E1 alpha subunit (368 amino acids, Mr 41,312, without the initiating methionine residue) and E1 beta subunit (324 amino acids, Mr 35,306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1 beta gene begins just 3 bp downstream from the E1 alpha stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The E1 alpha and E1 beta subunits of the B. stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1 alpha and E1 beta subunits of pyruvate and branched-chain 2-oxo-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the E1 alpha chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.

Acetyltransferases↗
PubMed1989

Reductive acetylation of tandemly repeated lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli is random order.

In vitro deletion and site-directed mutagenesis of the aceF gene of Escherichia coli was used to generate dihydrolipoamide acetyltransferase (E2p) polypeptide chains containing various permutations and combinations of functional and non-functional lipoyl domains. A lipoyl domain was rendered non-functional by converting the lipoylatable lysine residue to glutamine. Two- and three-lipoyl domain E2p chains, with lipoyl-lysine (Lys244) substituted by glutamine in the innermost lipoyl domains (designated +/- and +/+/-, respectively), and similar chains with lipoyl-lysine (Lys143) substituted by glutamine in the outer lipoyl domains (designated -/+ and -/-/+), were constructed. In all instances, pyruvate dehydrogenase complexes were assembled in vivo around E2p cores composed of the modified peptide chains. All the complexes were essentially fully active in catalysis, although the complex containing the -/-/+ version of the E2p polypeptide chain showed a 50% reduction in specific catalytic activity. Similarly, active-site coupling in the complexes containing the +/-, +/+/- and -/+ constructions of the E2p chains was not significantly different from that achieved by the wild-type complex. However, active-site coupling in the complex containing the -/-/+ version of the E2p chain was slightly impaired, consistent with the reduced overall complex activity. These results indicate that during oxidative decarboxylation there is no mandatory order of reductive acetylation of repeated lipoyl domains within E2p polypeptide chains, and strongly suggest that the three tandemly repeated lipoyl domains in the wild-type E2p chain function independently in the pyruvate dehydrogenase complex.

Acetylation↗
PubMed1976

Fatal lactic acidosis in a newborn attributable to a congenital defect of pyruvate dehydrogenase.

An infant suffering from metabolic acidosis attributable to hyperlactatemai (6.1 mmol/liter) accompanied by hyperalaninemia (1 mmol/liter) and hyperserinemia (0.6 mmol/liter) is described. The urinary excretion of lactate and pyruvate was greatly elevated; the lactate to pyruvate ratio was normal. The urine showed low levels of citrate, isocitrate, and cis-aconitate, and low or normal levels of alpha-oxoglutarate, succinate, malate, and methylmalonate. Aspartate was slightly elevated in serum and urine, indicating a corresponding increase if its alpha-ketoacid oxaloacetatae. These patterns of organic acids and amino acids suggested an in vivo defect in the oxidation of pyruvate. Fibroblasts cultured from skin biopsy from the patient metabolized radioactive pyruvate (final concentration 0.04-2 mmol/liter) to CO2 at rates from 5 to 17% of that of fibroblasts from normal control subjects. Enzyme studies with fibroblast sonicates revealed a severe deficiency of the pyruvate dehydrogenase complex (about 8% of normal), and this error was localized to the first unit of the complex, i.e., the pyruvate dehydrogenase (about 4% of normal). Fibroblasts from both parents metabolized pyruvate to CO2 at a slightly reduced rate, suggesting parental heterozygosity.

Acidosis↗
PubMed1989

2-Acetylthiamin pyrophosphate: an enzyme-bound intermediate in thiamin pyrophosphate-dependent reactions.

Several enzymes catalyze reactions that may involve acetylthiamin pyrophosphate (acetyl-TPP) as an intermediate. These enzymes are phosphoketolase, pyruvate oxidase and several pyruvate oxidoreductases. Acetyl-TPP can be synthesized and used as a carrier to analyze quenched reaction mixtures for the presence of [14C]acetyl-TPP. Synthetic acetyl-TPP exhibits unusual chemical properties and a unique pH-rate profile that serves as a powerful means of characterizing [14C]acetyl-TPP that has been isolated from quenched enzymatic reaction mixtures. Using this and other methods, extensive evidence has been obtained for the involvement of acetyl-TPP in certain reactions catalyzed by the pyruvate dehydrogenase complex (PDH complex) of Escherichia coli. Acetyl-TPP is chemically competent as an intermediate in the decarboxylation and dehydrogenation of pyruvate by the PDH complex; and it is transiently formed during the course of this reaction. It may be an enzyme-bound intermediate or it may be in equilibrium with such an intermediate. Acetyl-TPP is very likely to be an intermediate of the phosphoketolase reaction. However, no direct evidence linking it to the phosphoketolase reaction mechanism is yet available. It is unclear whether acetyl-TPP is an intermediate in the pyruvate oxidoreductase reactions. In one example, that of the ketoacid oxidoreductase of Halobacterium halobium, analysis by electron paramagnetic resonance spectroscopy indicates the involvement of a hydroxyethyl-TPP-radical as an intermediate. It is unknown whether the subsequent reaction of this radical with coenzyme A an an oxidized FeS cluster to produce acetyl coenzyme A and the reduced cluster involves the intermediate formation of acetyl-TPP.

Aldehyde-Lyases↗
PubMed1978

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The intramolecular passage of substrate between the component enzymes of the pyruvate dehydrogenase multienzyme complex of Escherichia coli was examined. A series of partly reassembled complexes, varying only in their E1 (pyruvate decarboxylase, EC 1.2.4.1) content, was incubated with pyruvate in the absence of CoA, conditions under which the lipoic acid residues covalently bound to the E2 (lipoate acetyltransferase, EC2.3.1.12) chains of the complex become reductively acetylated, and the reaction then ceases. The fraction of E2 chains thus acetylated was estimated by specific reaction of the thiol groups in the acetyl-lipoic acid moieties with N-ethyl[2,3-14C]maleimide. The simplest interpretation of the results was that a single E1 dimer is capable of catalysing the rapid acetylation of 8-12 E2 chains, in good agreement with the results of Bates, Danson, Hale, Hooper & Perham [(1977) Nature (London) 268, 313-316]. This novel functional connexion of active sites must be brought about by transacetylation reactions between lipoic acid residues of neighbouring E2 chains in the enzyme complex. There was also a slow transacylation process between the rapidly acetylated lipoic acid residues and those that did not react in the initial, faster phase. This interaction was not investigated in detail, since it is too slow to be of kinetic significance in the normal enzymic reaction.

Binding Sites↗
PubMed1981

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complexes from bacterial and mammalian sources.

A simple method was developed for assessing the intramolecular coupling of active sites in the lipoate acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complexes from Escherichia coli, Bacillus stearothermophilus and ox heart and pig heart mitochondria. Samples of enzyme complex were prepared in which the pyruvate decarboxylase (E1) component was selectively and partly inhibited by treatment with increasing amounts of a transition-state analogue, thiamin thio-thiazolone pyrophosphate. The fraction of the E2 component acetylated by incubation with [2-14C] pyruvate, in the absence of CoA, was determined for each sample of partly inhibited enzyme and was found in all cases to exceed the fraction of overall complex activity remaining. This indicated the potential for transacetylation reactions among the lipoic acid residues within the E2 core. A graphic presentation of the data allowed comparison of the active-site coupling in the various enzymes, which may differ in their lipoic acid content (one or two residues per E2 chain). It is clear that active-site coupling is a general property of pyruvate dehydrogenase complexes of octahedral and icosahedral symmetries, the large numbers of subunits in each E2 core enhancing the effect.

Acetylation↗
PubMed1991

An improved assay for pyruvate dehydrogenase in liver and heart.

A radiochemical assay was developed to measure pyruvate dehydrogenase complex (PDC) activity in liver and heart without interference by branched-chain 2-oxo acid dehydrogenase (BCODH). Decarboxylation of pyruvate by BCODH was eliminated by using low pyruvate concentration (0.5 mM), a preferred substrate for BCODH (3-methyl-2-oxopentanoate) that is not used by PDC, and a competitive inhibitor of BCODH, dichloroacetate. This method was validated by assaying a combination of both purified enzymes and tissue homogenates with known amounts of added BCODH. The actual percentage of active PDC decreased after 48 h starvation from 13.6 to 3.1 in liver and from 77.1 to 9.0 in heart. Total PDC activity (munits of PDC/units of citrate synthase) in starved rats was increased by 34% in liver and decreased by 23% in heart. Total PDC activity (munits/g wet wt.) in fed- and starved-rat liver was 0.8 and 1.3, and in heart was 6.6 and 5.8, respectively.

Animals↗
PubMed1986

Phosphorylation-dephosphorylation of pyruvate dehydrogenase from bakers' yeast.

The pyruvate dehydrogenase complex was purified to homogeneity from bakers' yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase kinase activity was detected at any stage of the purification. However, the purified pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. The protein-bound radioactivity was localized in the pyruvate dehydrogenase alpha subunit. The phosphorylated, inactive pyruvate dehydrogenase complex was dephosphorylated and reactivated with purified pyruvate dehydrogenase phosphatase from bovine heart. Tryptic digestion of the 32P-labeled complex yielded a single phosphopeptide, which was purified to homogeneity. The sequence of the phosphopeptide was established to be Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphotetradecapeptide derived from the alpha subunit of bovine kidney and heart pyruvate dehydrogenase: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.

Adenosine Triphosphate↗
PubMed1994

Loss of fatty acid control of gluconeogenesis and PDH complex flux in adrenalectomized rats.

This work aimed to determine the role played by the adrenal gland in the fatty acid control of gluconeogenesis in isolated perfused rat livers. The gluconeogenic substrate concentration responses were not altered in adrenalectomized (ADX) rats. This observation indicates that glucocorticoids are not essential to maintain normal basal gluconeogenic rates. In contrast, fatty acid failed to stimulate gluconeogenesis from lactate and elicited attenuated stimulation with pyruvate as substrate in livers from ADX rats. Fatty acid-induced stimulation of respiration and ketone body production were similar in control and ADX rats. Thus the diminished responsiveness of the gluconeogenic pathway to fatty acid cannot be the result of different rates of energy production and/or generation of reducing power. Fatty acids did not inhibit pyruvate decarboxylation in livers from ADX rats. Even though mitochondria isolated from livers of ADX rats showed normal basal rates of pyruvate metabolism, fatty acids failed to inhibit pyruvate decarboxylation and the activity of the pyruvate dehydrogenase complex. This novel observation of the glucocorticoid effect in controlling the pyruvate dehydrogenase complex responsiveness indicates that the mitochondrial partitioning of pyruvate between carboxylation and decarboxylation reactions may be altered in livers from ADX rats. We propose that the diminished effect of fatty acid in stimulating gluconeogenesis in livers from ADX rats is the result of a limited pyruvate availability for the carboxylase reaction due to a lack of inhibition of flux through the pyruvate dehydrogenase complex.

Adrenalectomy↗
PubMed1990

Regulation of the branched-chain alpha-ketoacid dehydrogenase and elucidation of a molecular basis for maple syrup urine disease.

The hepatic branched-chain alpha-ketoacid dehydrogenase complex plays an important role in regulating branched-chain amino acid levels. These compounds are essential for protein synthesis but toxic if present in excess. When dietary protein is deficient, the hepatic enzyme is converted to the inactive, phosphorylated state to conserve branched-chain amino acids for protein synthesis. When dietary protein is excessive, the enzyme is in the active, dephosphorylated state to commit the excess branched-chain amino acids to degradation. Inhibition of protein synthesis by cycloheximide, even when the animal is starving for dietary protein, results in activation of the hepatic branched-chain alpha-ketoacid dehydrogenase complex to prevent accumulation of branched-chain amino acids. Likewise, the increase in branched-chain amino acids caused by body wasting during starvation and uncontrolled diabetes is blunted by activation of the hepatic branched-chain alpha-ketoacid dehydrogenase complex. The activity state of the complex is regulated in the short term by the concentration of branched-chain alpha-ketoacids (inhibitors of branched-chain alpha-ketoacid dehydrogenase kinase) and in the long term by alteration in total branched-chain alpha-ketoacid dehydrogenase kinase activity. cDNAs have been cloned and the primary structure of the mature proteins deduced for the E1 alpha subunit of the human and rat liver branched-chain alpha-ketoacid dehydrogenase complex. The cDNA and protein sequences are highly conserved for the two species. Considerable sequence similarity is also apparent between the E1 alpha subunits of the human branched-chain alpha-ketoacid dehydrogenase complex and the pyruvate dehydrogenase complex. Maple syrup urine disease is caused by an inherited deficiency in the branched-chain alpha-ketoacid dehydrogenase complex. The molecular basis of one maple syrup urine disease family has been determined for the first time. The patient was found to be a compound heterozygote, inheriting an allele encoding an abnormal E1 alpha from the father, and an allele which is not expressed from the mother. The only known animal model for the disease (Polled Hereford cattle) has also been characterized. The mutation in these animals introduces a stop codon in the leader peptide of the E1 alpha subunit, resulting in premature termination of translation. Two thiamine responsive patients have been studied. The deduced amino acid sequences of the mature E1 alpha subunit and its leader sequence were normal, suggesting that the defect in these patients must exist in some other subunit of the complex. 3-Hydroxyisobutyrate dehydrogenase and methylmalonate-semialdehyde dehydrogenase, two enzymes of the valine catabolic pathway, were purified from liver tissue and characterized.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMedDate not supplied

Pyruvate dehydrogenase deficiency: clinical and biochemical diagnosis.

A female neonate with pyruvate dehydrogenase (PDH) deficiency is presented with clinical, radiologic, biochemical, neuropathologic, and molecular genetic data. She was dysmorphic, with a high forehead, lowset ears, thin upper lip, upturned nose, and rhizomelic limbs. Cranial MRI revealed severe cortical atrophy, ventricular dilatation, and corpus callosum agenesis. Pyruvate and lactate levels were increased in CSF and blood. Urinary organic acid profile was compatible with PDH deficiency. PDH activity was normal in fibroblasts, lymphocytes, and muscle. The PDH E1-alpha gene was sequenced and a single base mutation was found within the regulatory phosphorylation site in exon 10. It is postulated that this mutation causes a cerebral form of PDH deficiency. Tissue-specific expression of the disease could be explained by differential X chromosome inactivation because the PDH E1-alpha gene is located on this chromosome. Dysmorphism with severe cerebral malformations in female patients merits a metabolic evaluation, including determination of lactate and pyruvate levels in CSF.

Brain↗
PubMed1999

Cerebrospinal fluid levels of thiamine in patients with Parkinson's disease.

Thiamine is an essential cofactor for several important enzymes involved in brain oxidative metabolism, such as the alpha-ketoglutarate dehydrogenase complex (KGDHC), pyruvate-dehydrogenase complex, and transketolase. The activity of KGDHC is decreased in the substantia nigra or patients with Parkinson's disease (PD). We measured cerebrospinal (CSF) levels of thiamine-diphosphate, thiamine-monophosphate, free thiamine, and total thiamine, using ion-pair reversed phase high performance liquid chromatography, in 24 PD patients and 40 matched controls. The mean CSF levels of thiamine-derivatives did not differ significantly from those of controls, with the exception of lower CSF free thiamine levels in the PD-patient group. PD patients under levodopa therapy had significantly higher CSF thiaminediphosphate and total thiamine than those not treated with this drug. CSF thiamine levels were not correlated with age, age at onset, duration of the disease, scores of the Unified Parkinson Disease Rating Scale of the Hoehn and Yahr staging in the PD group. These results suggest that low CSF free thiamine levels could be related with the risk for PD.

Antiparkinson Agents↗