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PubMed1982

Cross-linking and 1H n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Escherichia coli.

The pyruvate dehydrogenase complex of Escherichia coli was treated with o-phenylene bismaleimide in the presence of the substrate pyruvate, producing almost complete cross-linking of the lipoate acetyltransferase polypeptide chains as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This took place without effect on the catalytic activities of the other two component enzymes and with little evidence of cross-links being formed with other types of protein subunit. Limited proteolysis with trypsin indicated that the cross-links were largely confined to the lipoyl domains of the lipoate acetyltransferase component of the same enzyme particle. This intramolecular cross-linking had no effect on the very sharp resonances observed in the (1)H n.m.r. spectrum of the enzyme complex, which derive from regions of highly mobile polypeptide chain in the lipoyl domains. Comparison of the spin-spin relaxation times, T(2), with the measured linewidths supported the idea that the highly mobile region is best characterized as a random coil. Intensity measurements in spin-echo spectra showed that it comprises a significant proportion (probably not less than one-third) of a lipoyl domain and is thus much more than a small hinge region, but there was insufficient intensity in the resonances to account for the whole lipoyl domain. On the other hand, no evidence was found in the (1)H n.m.r. spectrum for a substantial structured region around the lipoyl-lysine residues that was free to move on the end of this highly flexible connection. If such a structured region were bound to other parts of the enzyme complex for a major part of its time, its resonances might be broadened sufficiently to evade detection by (1)H n.m.r. spectroscopy.

Cross-Linking Reagents↗

PubMed1995

An investigation of the kinase activity of aurochs heart pyruvate dehydrogenase complex.

The purified aurochs heart pyruvate dehydrogenase complex (PDC) saturated to approximation 60% with endogenous thiamine pyrophosphate (TPP), was slowly and incompletely inactivated by its kinase in the presence of ATP. Exogenous TPP or ADP, but not pyruvate, strongly inhibited the kinase activity. The kinetic properties of the aurochs heart PDC kinase suggested the occurrence of two active sites each with different affinities for ATP (K'm - 1.7 microM, K''m = 48 microM).

Adenosine Triphosphate↗

PubMed1990

Molecular biology and biochemistry of pyruvate dehydrogenase complexes.

In most organisms, the pyruvate dehydrogenase complex catalyzes the pivotal irreversible reaction that leads to the consumption of glucose in the aerobic, energy-generating pathways. A combination of biochemical and molecular biology studies have greatly expanded our understanding of the overall structural organization of this multicomponent system, delineated the locations and elucidated the functions of structural domains of the catalytic components, and revealed significant evolutionary changes. Important to this progress was the deduction of the primary amino acid sequences from cDNA clones for each of the catalytic components from several species. The greatest detail is available for the FAD-containing dihydrolipoamide dehydrogenase component, which is the only component for which tertiary structure information has recently emerged. For the dihydrolipoamide acetyltransferase core component, a similar but species-variable multidomain structure is established that is responsible for the distinct architectures of the inner cores, the peripheral binding of the other components, and the conveyance of reaction intermediates between distantly separated active sites. A second lipoyl-bearing component, protein X, has been shown to play a critical role in the organization and function of the complex from many higher organisms. Although much is known about the means of effector modulation of mammalian complex activity, identification of the signal eliciting its regulation by insulin still poses an exciting challenge.

Amino Acid Sequence↗

PubMed1989

Familial intermittent ataxia due to a defect of the E1 component of pyruvate dehydrogenase complex.

Disturbances of pyruvate metabolism have been implicated in the aetiology of several neurological disorders including Leigh's disease and familial ataxia. We have re-investigated a patient whose initial description documented intermittent ataxia, a presumed disorder of pyruvate metabolism and an X-linked pattern of inheritance. Recent studies showed he had slow oxidation of pyruvate, low pyruvate dehydrogenase complex (PDC) activity and immunochemical evidence of E1 deficiency in skeletal muscle mitochondria. This is consistent with the recent finding that the gene for E1 alpha is on the X chromosome.

Adult↗

PubMed1986

Inhibition of pyruvate dehydrogenase complex by moniliformin.

The mechanism for the inhibition of pyruvate dehydrogenase complex from bovine heart by moniliformin was investigated. Thiamin pyrophosphate proved to be necessary for the inhibitory action of moniliformin. The inhibition reaction was shown to be time-dependent and to follow first-order and saturation kinetics. Pyruvate protected the pyruvate dehydrogenase complex against moniliformin inactivation. Extensive dialysis of the moniliformin-inactivated complex only partially reversed inactivation. Moniliformin seems to act by inhibition of the pyruvate dehydrogenase component of the enzyme complex and not by acting on the dihydrolipoamide transacetylase or dehydrogenase components, as shown by monitoring the effect of moniliformin on each component individually. On the basis of these results, a suicide inactivator mechanism for moniliformin on pyruvate dehydrogenase is proposed.

Cyclobutanes↗

PubMed1979

Amino acid sequences around the sites of phosphorylation in the pig heart pyruvate dehydrogenase complex.

1. When pig heart pyruvate dehydrogenase complex was phosphorylated to completion with [gamma-32P]ATP by its intrinsic kinase, three phosphorylation sites were observed. The amino acid sequences around these sites were: sequence 1, Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg; and sequence 2, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser(P)-Tyr-Arg. 2. When phosphorylated to inactivation by repetitive additions of limiting quantities of [gamma-32P]ATP, phosphate was incorporated mainly (more than 90%) into Ser-5 of sequence 2. Phosphorylation of this site thus results in activation of pyruvate dehydrogenase. 3. If Ser-5 is phosphorylated with ATP and the enzyme then incubated with [gamma-32P]ATP, phosphorylation of the remaining sites occurred. Ser-12 of sequence 2 is phosphorylated about twice as rapidly as Ser-6 of sequence 1. 4. Incubation of pyruvate dehydrogenase with excess [gamma-32P]ATP with termination of phosphorylation at about 50% complete inactivation showed that Ser-5 of sequence 2 was phosphorylated most rapidly, but also that Ser-12 of sequence 2 was significantly (15% of total) phosphorylated. Ser-6 sequence 1 contained about 1% total P. 5. These results suggest that addition of limiting amounts of ATP produces primarily phosphorylation of Ser-5 of sequence 2 (inactivating site). This also occurs during incubation with excess ATP before complete inactivation occurs, but a greater occupancy of other sites also occurs during this treatment.

Amino Acid Sequence↗

PubMed2006

Structural insight into interactions between dihydrolipoamide dehydrogenase (E3) and E3 binding protein of human pyruvate dehydrogenase complex.

The 9.5 MDa human pyruvate dehydrogenase complex (PDC) utilizes the specific dihydrolipoamide dehydrogenase (E3) binding protein (E3BP) to tether the essential E3 component to the 60-meric core of the complex. Here, we report crystal structures of the binding domain (E3BD) of human E3BP alone and in complex with human E3 at 1.6 angstroms and 2.2 angstroms, respectively. The latter structure shows that residues from E3BD contact E3 across its 2-fold axis, resulting in one E3BD binding site on the E3 homodimer. Negligible conformational changes occur in E3BD upon its high-affinity binding to E3. Modifications of E3BD residues at the center of the E3BD/E3 interface impede E3 binding far more severely than those of residues on the periphery, validating the "hot spot" paradigm for protein interactions. A cluster of disease-causing E3 mutations located near the center of the E3BD/E3 interface prevents the efficient recruitment of these E3 variants by E3BP into the PDC, leading to the dysfunction of the PDC catalytic machine.

Amino Acid Sequence↗

PubMed1998

Pyruvate dehydrogenase complex deficiency and absence of subunit X.

The pyruvate dehydrogenase complex (PDHc) is a multienzyme complex consisting of three catalytic and two regulatory enzymes, as well as a less well defined subunit called protein X. PDHc deficiency is a common cause of congenital lactic acidosis. Most patients with PDH deficiency have a mutation in the alpha chain of the PDH E1 enzyme. Very few patients have been described in whom the basic defect of a PDH deficiency is situated in the X protein. We studied a boy with severe lactic acidosis and developmental delay in whom a deficiency of PDH activity led to further investigations. Immunochemical analysis with anti-PDHc antibodies demonstrated an absence of the X component. This report is the fourth family in which an abnormal protein X has been found. In cases with PDH deficiency where no mutation of the PDHE1 alpha gene is found, further investigations by means of immunoblotting with specific antibodies against the different subunits should be performed.

Female↗

PubMed1977

Structure of pyruvate dehydrogenase complex. Comparison between freeze-etching and negative staining.

Pyruvate dehydrogenase complex (pyruvate : lipoate oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1), from pig heart, was studied by spray freeze etching and negative staining. From freeze etching experiments an average particle weight of 7-10(6) was estimated. Negative staining after glutaraldehyde fixation and freeze etching of unfixed and prefixed enzyme solutions yielded no significant difference in particle dimensions: the majority of the isometric complex molecules measured approximately 400 A in diameter. Tantalum tungsten shadowed freeze etch replicas indicated that the surface of the complex is built up of globular units. The relative positions of these units are in good agreement with the model still under discussion.

Animals↗

PubMed1981

[Lag period as affected by the adrenal pyruvate dehydrogenase complex containing residual endogenous thiamine pyrophosphate].

The pyruvate dehydrogenase complex from bovine adrenals does not completely lose thiamine pyrophosphate (TPP) in the course of purification. The preparations contain firmly bound residual TPP in the amount providing for one third of the maximal activity of the complex. A lag period (tau) is observed during catalysis by the pyruvate dehydrogenase complex with the residual TPP in the absence of exogenous coenzyme. The duration of the lag period depends on concentration of the complex: tau is decreased as the concentration increases. The decrease of the lag period occurs after preincubation of the complex with pyruvate, but it is the most remarkable after preincubation with pyruvate and CoA. The tetrahydrothiamine pyrophosphate at low concentrations completely inhibits the activity. However, the residual TPP is retained as is evidenced by the recovery of the complex activity after gel-filtration through Sephadex G-25. The development of the lag period may be interpreted in terms of partial limited dissociation of the residual TPP in the region of the catalytic center.

Adrenal Glands↗

PubMed1981

Pyruvate dehydrogenase complex activity in normal and deficient fibroblasts.

Pyruvate dehydrogenase complex (PDC) activity in human skin fibroblasts appears to be regulated by a phosphorylation-dephosphorylation mechanism, as is the case with other animal cells. The enzyme can be activated by pretreating the cells with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, before they are disrupted for measurement of PDC activity. With such treatment, the activity reaches 5-6 nmol/min per mg of protein at 37 degrees C with fibroblasts from infants. Such values represent an activation of about 5-20-fold over those observed with untreated cells. That this assay, based on [1-(14)C]pyruvate decarboxylation, represents a valid measurement of the overall PDC reaction is shown by the dependence of (14)CO(2) production on the presence of thiamin-PP, coenzyme A (CoA), Mg(++), and NAD(+). Also, it has been shown that acetyl-CoA and (14)CO(2) are formed in a 1:1 ratio. A similar degree of activation of PDC can also be achieved by adding purified pyruvate dehydrogenase phosphatase and high concentrations of Mg(++) and Ca(++), or in some cases by adding the metal ions alone to the cell homogenate after disruption. These results strongly suggest that activation is due to dephosphorylation. Addition of NaF, which inhibits dephosphorylation, leads to almost complete loss of PDC activity. Assays of completely activated PDC were performed on two cell lines originating from patients reported to be deficient in this enzyme (Blass, J. P., J. Avigan, and B. W. Ublendorf. 1970. J. Clin. Invest. 49: 423-432; Blass, J. P., J. D. Schuman, D. S. Young, and E. Ham. 1972. J. Clin. Invest. 51: 1545-1551). Even after activation with DCA, fibroblasts from the patients showed values of only 0.1 and 0.3 nmol/min per mg of protein. A familial study of one of these patients showed that both parents exhibited activity in fully activated cells about half that of normal values, whereas cells from a sibling appeared normal. These results demonstrate the inheritance nature of PDC deficiency, and that the present assay is sufficient to detect the heterozygous carriers of the deficiency. Application of the same procedures to fibroblasts obtained from 16 individuals who were believed to have normal PDC activities showed a range from about 2-2.5 nmol/min per mg protein for adults to 5-6 nmol/min per mg protein for cells from infants.

Acetyl Coenzyme A↗

PubMed2004

Optimized spectrophotometric assay for the completely activated pyruvate dehydrogenase complex in fibroblasts.

BACKGROUND: Analysis of the pyruvate dehydrogenase complex (PDHc) activity in human skin fibroblasts is hampered by low enzyme activity in the cells. The most commonly used radiochemical method detects the formation of (14)CO(2), an endproduct of the E1 component of PDHc, from [1-(14)C]pyruvate. METHODS: We report a spectrophotometric method for the analysis of PDHc activity in fibroblasts based on detection of NADH formation via a p-iodonitrotetrazolium violet (INT)-coupled system. We investigated in detail the specific requirements of this assay, such as cofactor requirements and the effects of suggested stimulatory compounds and different cell disruption procedures. The reliability of the optimized assay was studied by investigation of patients previously diagnosed with PDHc deficiency and by comparison with results from the radiochemical method. RESULTS: Mean (SD) total PDHc activities were 136 (31) and 58 (21) mU/U of citrate synthase in fibroblast homogenates from 10 healthy volunteers and 7 PDHc-deficient patients, respectively, by the spectrophotometric assay. Similar results were obtained in a mitochondrial fraction. Dithiothreitol (DTT) increased the nonspecific inhibitor-insensitive rate with less pronounced effect on the specific rate of PDHc activity. Administration of DTT increased PDHc activity to 193 (3)% of control activity (without DTT), but decreased the inhibitor-sensitive rate from 99 (0.3)% (without DTT) to 69 (2)% (with 0.3 mmol/L DTT). CONCLUSION: The simple, optimized spectrophotometric assay for PDHc analysis allows reliable investigation of the enzyme complex in human skin fibroblasts.

Cells, Cultured↗

PubMedDate not supplied

[Isolation and characterization of a membrane-bound pyruvate dehydrogenase complex from the phototrophic bacterium Rhodospirillum rubrum (author's transl)].

The pyruvate dehydrogenase complex from the photosynthetic bacterium Rhodospirillum rubrum was associated with the membrane fraction both in heterotrophically and photosynthetically grown cells. The complex was separated from the membranes and partially purified by precipitation with MgSO4 and gelfiltration through Sepharose 4B. The purified complex had a specific activity of 1.5-2mumol/min-mg protein and contained the following partial activities: pyruvate dehydrogenase (EC 1.2.4.1), dihydrolipoamide transacetylase (EC 2.3.1.12) and dihydrolipoamide dehydrogenase (EC 1.6.4.3). Contrary to other bacterial pyruvate dehydrogenase complexes, the enzyme complex from R. rubrum revealed no cooperatively between pyruvate binding sites. The kinetic constants (Km) for the overall reaction were (in mM): 0.14 (pyruvate), 0.07 (NAD) and 0.025 (coenzyme A). The Km for thiamine pyrophosphate was dependent on the nature and the concentration of the divalent metal ion (Mn or Mg) present in the reaction mixture, the values ranging from 0.5 to 3 micrometer. NADH was a potent inhibitor (Ki=5 micrometer) of the enzyme complex and the dihydrolipo amide dehydrogenase. The inhibition was competitive with respect to NAD. In addition to its rapid inhibitory effect, NADH also inactivated the enzyme. Cysteine partially protected the enzyme complex against NADH-inactivation. Acetyl-coenzyme A also inhibited the overall reaction (Ki=40 micrometer). The inhibition was dependent on the concentration of coenzyme A, but independent of the concentration of pyruvate. Sugar phosphates, phosphoenolpyruvate, citric acid cycle intermediates and nucleosidephosphates (1 mM) had no pronounced effect on the overall reaction.

Acetyl Coenzyme A↗

PubMed1999

Characterization of the dihydrolipoamide acetyltransferase of the mitochondrial pyruvate dehydrogenase complex from potato and comparisons with similar enzymes in diverse plant species.

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) can be disassociated in 1 M NaCl and 0.1 M glycine into a large dihydrolipoamide acetyltransferase (E2) complex and smaller pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3) complexes. The E2 complex consists of 55 and 78-kDa polypeptides which are reversibly radiolabelled to a similar degree in the intact mPDC by [2-14C]pyruvate. Affinity-purified antibodies against the 55-kDa protein do not cross-react with the 78-kDa protein and the two proteins show different peptide patterns following partial proteolysis. The 78 and 55-kDa proteins are present in approximately equal abundance in the E2 complex and incorporate a similar amount of [14C] on incubation with [2-14C]pyruvate. Native mPDC and the E2 complex have sedimentation coefficients of 50S and 30S, respectively. Titration of electro-eluted polypeptides against the intact mPDC and E2 complex revealed that each mg of mPDC contains 0.4 mg of E1, 0.4 mg of E2 and 0.2 mg of E3. Labelling of partially purified mPDC from potato, pea, cauliflower, maize and barley, with [2-14C]pyruvate, suggest that a 78-kDa acetylatable protein is only found in the dicotyledonous species, while all plant species tested contained a smaller 52-60 kDa acetylatable protein.

Acetylation↗

PubMed1983

The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the pyruvate dehydrogenase component.

The nucleotide sequence of a 3780-base-pair segment of DNA containing the aceE gene encoding the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The aceE structural gene comprises 2655 base pairs (885 codons, excluding the initiation codon AUG), it is preceded by a good ribosome binding site and several potential RNA polymerase binding sites. Its polarity and location in the restriction map of the corresponding segment of DNA are consistent with it being the proximal gene in the ace operon, as defined in previous genetic and post-infection labelling studies. The relative molecular mass (99474), composition (885 amino acids), amino-terminal residue and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with published information obtained from studies with the purified pyruvate dehydrogenase component (E1). The nucleotide sequence also contains a second gene (gene A) situated upstream of the aceE gene. It appears to be an independent gene containing 708 base pairs (236 codons) and encoding a weakly expressed product (protein A; Mr = 27049) of unknown function.

Amino Acids↗

PubMed1989

Subunit associations in the mammalian pyruvate dehydrogenase complex. Structure and role of protein X and the pyruvate dehydrogenase component binding domain of the dihydrolipoyl transacetylase component.

We have further distinguished the structures and roles of the two lipoyl-bearing components of the pyruvate dehydrogenase complex, the dihydrolipoyl transacetylase (E2) component and the component designated as protein X. The amino acid sequences of the NH2-terminal regions of the lipoyl-bearing domain of the E2 component and protein X are different but related. The dihydrolipoyl dehydrogenase (E3) component but not the pyruvate dehydrogenase (E1) component protected protein X against proteolytic degradation by trypsin and protease Arg C. Protein X-specific polyclonal antibodies inhibit reconstitution of the overall reaction catalyzed by the complex (E2-X subcomplex recombined with the E1 and E3 components). The rate of development of this inhibition was reduced by pretreatment of E2-X subcomplex with the E3 component. These data strongly suggest the E3 component associates with protein X. The E1 component (an alpha 2 beta 2 tetramer), but not the E3 component, reduced trypsin cleavage of E2 subunits at 4 degrees C and altered the patterns of cleavage at 22 degrees C. At 22 degrees C a large (Mr congruent to 49,000) outer domain (E2LB) of the E2 component was produced. E2LB had the same NH2-terminal amino acid sequence as the smaller (Mr congruent to 38,000) lipoyl-bearing domain (E2L). E2LB, in contrast to E2L, interacted with both the E1 component and the beta subunit of the E1 component. Thus the E1 component is bound through an E1-binding domain that is located in E2 subunits between the inner domain and the outer, lipoyl-bearing domain.

Acetyltransferases↗

PubMed1983

A study on the physical interaction between the pyruvate dehydrogenase complex and citrate synthase.

In this paper, physicochemical evidence is given for the association between the pyruvate dehydrogenase complex (EC 1.2.4.1) and citrate synthase (EC 4.1.3.7) with two gel chromatographic techniques with poly(ethylene glycol) co-precipitation and with ultracentrifugation. Experiments with active enzyme gel chromatography indicate that citrate synthase also associates with pyruvate dehydrogenase complex in its functioning state. Citrate synthase binds to the isolated transacetylase core of pyruvate dehydrogenase complex, but in the binding to the whole pyruvate dehydrogenase complex the two other components of the complex are also involved. One pyruvate dehydrogenase complex can bind 10-11 citrate synthase dimers, and the dissociation constant is about 5.7-6.0 microM as determined by two independent methods. The association between the pyruvate dehydrogenase complex and citrate synthase raises the possibility of the dynamic compartmentation of acetyl-CoA in the mitochondria which results in the direction of acetyl-CoA from pyruvate towards citrate.

Animals↗

PubMed1990

Primary biliary cirrhosis. Inhibition of pyruvate dehydrogenase complex activity by autoantibodies specific for E1 alpha, a non-lipoic acid containing mitochondrial enzyme.

Antimitochondrial antibodies (AMA) are serologically characteristic of patients with PBC. Four Ag recognized by AMA have been recently identified, including protein X and the acyltransferases of three related multienzyme complexes: the pyruvate dehydrogenase complex (PDC), the branched-chain alpha-ketoacid dehydrogenase complex, and the alpha-ketoglutarate dehydrogenase complex. Each of these enzymes contains one or more lipoyl moieties as part of a major functional site. In addition, epitope mapping has suggested that the AMA target is this lipoic acid-binding region. In this report we demonstrate that sera from patients with PBC also recognize the E1 component (pyruvate dehydrogenase, EC 1.2.4.1) of PDC. PDC-E1 is composed of alpha and beta-chains. Reactivity with the 41 kD alpha chain was detected in 80 of 120 (66%) PBC sera by immunoblotting against purified PDC-E1; 2 of 120 sera also demonstrated reactivity with the 34 kD beta chain. In contrast, sera from patients with SLE, chronic active hepatitis, or progressive sclerosing cholangitis as well as sera from healthy volunteers did not react with PDC-E1. Furthermore, affinity-purified PBC sera against PDC-E1 alpha were able to inhibit PDC enzyme activity, whereas control sera could not. PDC-E1 alpha is now the fifth mitochondrial autoantigen of PBC to be identified. Similar to the four previously identified autoantigens, AMA appear to be directed to a functional site of PDC-E1 alpha inasmuch as they are able to inhibit enzyme function. However, PDC-E1 alpha is also unique in that it is the first identified mitochondrial autoantigen which does not contain lipoic acid.

Antigen-Antibody Reactions↗