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PubMed2006

Molecular architecture of the pyruvate dehydrogenase complex: bridging the gap.

The PDC (pyruvate dehydrogenase complex) is a high-molecular-mass (4-11 MDa) complex of critical importance for glucose homoeostasis in mammals. Its multi-enzyme structure allows for substrate channelling and active-site coupling: sequential catalytic reactions proceed through the rapid transfer of intermediates between individual components and without diffusion into the bulk medium due to its 'swinging arm' that is able to visit all PDC active sites. Optimal positioning of individual components within this multi-subunit complex further affects the efficiency of the overall reaction and stability of its intermediates. Mammalian PDC comprises a 60-meric pentagonal dodecahedral dihydrolipoamide (E2) core attached to which are 30 pyruvate decarboxylase (E1) heterotetramers and six dihydrolipoamide (E3) homodimers at maximal occupancy. Stable E3 integration is mediated by an accessory E3-binding protein associated with the E2 core. Association of the peripheral E1 and E3 enzymes with the PDC core has been studied intensively in recent years and has yielded some interesting and substantial differences when compared with prokaryotic PDCs.

Bacterial Proteins↗
PubMed1997

Pyruvate dehydrogenase complex deficiency with multiple minor anomalies.

Pyruvate dehydrogenase complex (PDHC) deficiency is known to cause congenital lactic acidosis. The case of a 9-month-old female infant with PDHC deficiency caused by a mutation in exon 11 of the pyruvate dehydrogenase (PDH) E1 alpha gene is described. Her facial features were as follows: frontal bossing, upslanting palpebral fissures, a short upturned nose, a long philtrum and low set ears. These anomalies are characteristic not only of a malformation syndrome or chromosomal aberration, but also of PDHC deficiency. Because PDHC deficiency requires early treatment, metabolic disorders should be kept in mind in a patient with dysmorphic features. Further, she had multiple minor anomalies including bilateral inguinal herniae, an umbilical hernia and small hands and feet, which have not been described in previous reports.

Abnormalities, Multiple↗
PubMed1983

Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex.

In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.

Animals↗
PubMed2003

Regulation of pyruvate dehydrogenase complex activity in plant cells.

The pyruvate dehydrogenase complex (PDC) is subjected to multiple interacting levels of control in plant cells. The first level is subcellular compartmentation. Plant cells are unique in having two distinct, spatially separated forms of the PDC; mitochondrial (mtPDC) and plastidial (plPDC). The mtPDC is the site of carbon entry into the tricarboxylic acid cycle, while the plPDC provides acetyl-CoA and NADH for de novo fatty acid biosynthesis. The second level of regulation of PDC activity is the control of gene expression. The genes encoding the subunits of the mt- and plPDCs are expressed following developmental programs, and are additionally subject to physiological and environmental cues. Thirdly, both the mt- and plPDCs are sensitive to product inhibition, and, potentially, to metabolite effectors. Finally, the two different forms of the complex are regulated by distinct organelle-specific mechanisms. Activity of the mtPDC is regulated by reversible phosphorylation catalyzed by intrinsic kinase and phosphatase components. An additional level of sensitivity is provided by metabolite control of the kinase activity. The plPDC is not regulated by reversible phosphorylation. Instead, activity is controlled to a large extent by the physical environment that exists in the plastid stroma.

Gene Expression Regulation, Plant↗
PubMed1993

[The effect of phosphorylation on catalytic function of muscle pyruvate dehydrogenase complex].

It has been shown that phosphorylation of the pyruvate dehydrogenase complex from pigeon breast muscle by endogenous ATP-dependent protein kinase suppresses the substrate conversion in the pyruvate: acceptor oxidoreductase reactions and nonoxidative reactions monitored by pyruvate decline in the absence of CoA and NAD. To identify the catalytic step blocked by phosphorylation, CD spectroscopy was used which revealed the appearance and decay of the charge transfer complex between component E1 and thiamine pyrophosphate during the enzymatic reaction. Phosphorylation of the pyruvate dehydrogenase complex while lowering the affinity for thiamine pyrophosphate does not preclude the formation of holo-E1 but inhibits its interaction with pyruvate. Phosphorylated pyruvate dehydrogenase, like the dephosphorylated enzyme, reacts with 2-hydroxyethyl thiamine pyrophosphate in half of the active sites. In the presence of deacylating agents (CoA or dithiothreitol) all the sites are reactive. A conclusion is drawn that the alternating functioning of the active centers is preserved in reductive acetylation of the acceptor substrates by phospho-E1.

Animals↗
PubMed1987

Electron microscopic study on the size of pyruvate dehydrogenase complex in situ.

Isolated pig heart pyruvate dehydrogenase complex (PDC) has been reported to have a molecular mass of 8000 kDa (large PDC) and a diameter of about 45 nm. Studies were carried out to determine the size of PDC in situ. Active enzyme centrifugation showed that extracts of pig heart mitochondria contain, in addition to large (S20,w = 100-200 S) active complexes, catalytically active small PDC (S20,w = 30 S). In addition, small PDC (1000-3000 kDa) could be obtained by gel filtration of mitochondrial extract. If pure large PDC was chromatographed in Triton X-100, then a fraction of it appears in the 1000-3000-kDa range. Isolation of small PDC and rechromatography showed the formation of large PDC. Anti-PDC and ferritin-labeled second antibody were used in an attempt to determine the size of PDC in isolated inner membrane vesicles containing PDC and in permeabilized mitochondria. In both studies no large aggregates of ferritin particles were found which would correspond to the size of large PDC. The conclusion of these experiments is that PDC exists in situ in a smaller form than the isolated pure enzyme.

Animals↗
PubMed1982

Pig heart [35S]thiophosphoryl pyruvate dehydrogenase complexes.

Thiophosphorylation of the pig heart pyruvate dehydrogenase complex with ATP [35S] (adenosine 5'[gamma-thio]-triphosphate) was analogous to phosphorylation with [gamma-32P]ATP except that thiophosphorylation of sites 2 and 3 was more rapid. With pyruvate dehydrogenase phosphatase the rate of dethiophosphorylation was 0.5% of that of dephosphorylation. Thiophosphorylation of sites 2 and 3 in complex phosphorylated in site 1 reduced the rate of dephosphorylation of site 1.

Adenosine Triphosphate↗
PubMed1989

Regulation of pea mitochondrial pyruvate dehydrogenase complex : does photorespiratory ammonium influence mitochondrial carbon metabolism?

Inactivation of the pyruvate dehydrogenase complex catalyzed by pyruvate dehydrogenase kinase was studied using intact mitochondria purified from green leaf tissue of pea (Pisum sativum L.) and dialyzed mitochondrial extracts. Thiamine pyrophosphate was inhibitory in dialyzed extracts but not in intact mitochondria, except in the presence of high concentrations of Na(+). NH(4) (+), at concentrations as low as 20 micromolar, markedly stimulated inactivation in dialyzed extracts. K(+) in the range 1 to 10 millimolar also enhanced inactivation. In contrast, Na(+) was without affect at lower concentrations but was inhibitory at 10 to 100 millimolar levels. The effect of NH(4) (+) is discussed in relation to a possible regulatory interaction between photorespiratory NH(4) (+) production and the entry of carbon into the tricarboxylic acid cycle by way of the pyruvate dehydrogenase complex.

Journal Article↗
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↗
PubMed1981

Substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli.

The investigation of the substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli allows a description of the binding region of pyruvate. Substrate analogs with electronegative substitutions in the methyl group show a strong competitive inhibition of the overall reaction of the pyruvate dehydrogenase complex. The most efficient inhibitor is fluoropyruvate which has a more than 100-fold higher affinity for the enzyme than pyruvate (Ki = 1.4 x 10(-6) M) does. The affinity of alpha-keto acids decreases with increasing chain length. Branched chain alpha-keto acids are even less effective inhibitors (Ki = approximately 0.02 M). alpha-Ketobutyrate is the only alpha-keto acid which is able to substitute for pyruvate as a substrate in the overall reaction of the enzyme complex. The Km value (3 mM) is 10-fold greater than that for pyruvate. The steady state kinetics of the overall reaction of alpha-ketobutyrate exhibits the same cooperativity (nh = 1.9) as seen with pyruvate. Small modifications of the carbonyl or the carboxyl group of pyruvate prevent binding completely. Binding of pyruvate to the pyruvate dehydrogenase complex may thus require interaction with two independent electrophilic centers. The acceptance of the methyl group seems not so much due to lipophilic interactions as to a steric effect. The experiments were carried out with an enzyme which was purified by a modified procedure which is faster and more convenient than previous methods. The procedure is applicable up to 0.5 liter of crude extract.

Binding Sites↗
PubMed1999

Regulatory effect of thiamin pyrophosphate on pig heart pyruvate dehydrogenase complex.

The kinetic behavior of pig heart pyruvate dehydrogenase complex (PDC) containing bound endogenous thiamin pyrophosphate (TPP) was affected by exogenous TPP. In the absence of exogenous TPP, a lag phase of the PDC reaction was observed. TPP added to the PDC reaction medium containing Mg2+ led to a disappearance of the lag phase, inducing strong reduction of the Km value for pyruvate (from 76.7 to 19.0 microM) but a more moderate decrease of Km for CoA (from 12.2 to 4.3 microM) and Km for NAD+ (from 70.2 to 33.6 microM), with no considerable change in the maximum reaction rate. Likewise, thiamin monophosphate (TMP) decreased the Km value of PDC for pyruvate, but to a lesser extent (from 76.7 to 57.9 microM) than TPP. At the unsaturating level of pyruvate, the A50 values for TPP and TMP were 0.2 microM and 0.3 mM, respectively. This could mean that the effect of TPP on PDC was more specific. In addition, exogenous TPP changed the UV spectrum and lowered the fluorescence emission of the PDC containing bound endogenous TPP in its active sites. The data obtained suggest that TPP plays, in addition to its catalytic function, the important role of positive regulatory effector of pig heart PDC.

Adenosine Diphosphate↗
PubMed1987

Regulation of pea mitochondrial pyruvate dehydrogenase complex activity: inhibition of ATP-dependent inactivation.

In contrast to the pyruvate dehydrogenase complex (PDC) from animal mitochondria, our in situ and in vitro studies indicate that the ATP:ADP ratio has little or no effect in regulating the mitochondrial pyruvate dehydrogenase complex from green pea seedlings. Pyruvate was a competitive inhibitor of ATP-dependent inactivation (Ki = 59 microM), while the PDC had a Km for pyruvate of microM. Thiamine pyrophosphate, the coenzyme for the pyruvate dehydrogenase (PDH) component of the complex, did not inhibit ATP-dependent inactivation when used alone but it enhanced inhibition by pyruvate. As such, thiamine pyrophosphate was a competitive inhibitor (Ki = 130 nM) of ATP-dependent inactivation. A model is proposed for the pyruvate plus thiamine pyrophosphate inhibition of ATP-dependent inactivation of the pyruvate dehydrogenase complex in which pyruvate exerts its inhibition of inactivation by altering or protecting the protein substrate from phosphorylation and not by directly inhibiting PDH kinase.

Adenosine Diphosphate↗
PubMed2006

YIL042c and YOR090c encode the kinase and phosphatase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex.

In Saccharomyces cerevisiae the pyruvate dehydrogenase (PDH) complex is regulated by reversible phosphorylation of its Pda1p subunit. We here provide evidence that Pda1p is phosphorylated by the mitochondrial kinase Yil042cp. Deletion of YOR090c, encoding a putative mitochondrial phosphatase, results in a decreased PDH activity, indicating that Yor090cp acts as the corresponding PDH phosphatase. We demonstrate by means of blue native gel electrophoresis and tandem affinity purification that both enzymes are associated with the PDH complex.

Amino Acid Sequence↗
PubMedDate not supplied

Purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of Neurospora crassa mitochondria.

A simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of Neurospora crassa mitochondria is described. After fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. Covalent chromatography on thiol-activated Sepharose CL-4B allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2-oxoglutarate, whereas the pyruvate dehydrogenase complex activity was retained in the presence of pyruvate. The purified 2-oxoglutarate dehydrogenase complex showed 4 protein bands by electrophoresis under dissociating conditions with apparent molecular weights of 160,000, 56,200, 55,600, 52,600 and a Km value of 3.8 X 10(-4) M for 2-oxoglutarate. The purified pyruvate dehydrogenase complex showed 5 protein bands with apparent molecular weights of 160,000, 57,600, 55,600, 52,500 and 37,100 and a Km value of 3.2 X 10(-4) M for pyruvate.

Chemical Precipitation↗
PubMed1983

[Enzyme activity of thiamine pyrophosphate in the rat after oxythiamine administration].

Intraperitoneal injection of hydroxythiamine to rats (1 mmol per kg bw) resulted after 2-4 h in a more than 4-fold decrease in the activity of the oxoglutarate dehydrogenase complex, pyruvate dehydrogenase complex and NADP-dependent isocitrate dehydrogenase in adrenal mitochondria. Inhibition of hyaloplasmic transketolase, 6-phosphogluconate dehydrogenase and NADP-dependent malate dehydrogenase occurred later. Based on the correlation of the time course of enzymatic activity in the adrenals and the decreased concentration of 11-hydroxycorticosteroids in the blood the paramount role in the maintenance of the steroidogenesis among thiamine pyrophosphate-containing enzymes is assigned to the oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes.

11-Hydroxycorticosteroids↗
PubMed1995

A library of monoclonal antibodies to Escherichia coli K-12 pyruvate dehydrogenase complex. A biochemical analysis and their ability to inhibit the enzyme complex.

A library of monoclonal antibodies to K-12 Escherichia coli pyruvate dehydrogenase complex (PDHc) and its pyruvate decarboxylating (EC 1.2.4.1; E1) subunit is reported. 21 monoclonal antibodies were generated, and 20 were investigated, of which 9 were elicited to PDHc and 11 to pure E1 subunit; 19 were of the IgG1 isotype and one of the IgG3 isotype. According to an enzyme immunoassay, all 20 of the monoclonal antibodies bound the PDHc, and 17 bound the E1 subunit. According to Western blot analysis, 14 of the 19 monoclonal antibodies bound to the E1 subunit. The monoclonal antibodies inhibited PDHc from 0 to > 98%. The six monoclonal antibodies that displayed greater than 30% inhibition of E. coli PDHc were unable to inhibit porcine heart PDHc nor did they bind porcine heart PDHc according to dot blot analysis. Radiolabeling gave binding constants ranging from 5 to 10 x 10(8) M-1 on these six monoclonal antibodies, with greater than 80% of maximal inhibition achieved in less than 1 min. One of the six, 18A9, gave > 98% inhibition, required two antibodies/E1 subunit for maximum inhibition, and was shown to be a non-competitive inhibitor. Monoclonal antibody 15A9 was shown to counteract GTP-induced inhibition, while 1F2 influenced the conformation of E1, allowing two antibodies, which did not previously bind E1, to bind to it. A new mechanism-based kinetic assay is presented that is specific for the E1 component of 2-keto acid dehydrogenases. This assay confirmed that the three most strongly inhibitory monoclonal antibodies specifically inhibited the E1 function while antibody 1F2 led to enhanced activity, suggesting an induced conformational change in PDHc or in E1.

Animals↗