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PubMed1981

Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a bifunctional arsenoxide: selective inactivation of lipoamide dehydrogenase.

The bifunctional reagent p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH2CONHPhAsO) in the presence of excess reduced nicotinamide adenine dinucleotide has been shown to cause the irreversible active site directed inactivation of the lipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli. The ability of the lipoate acetyltransferase (E2) component to bind coenzyme A was decreased by about 50% in this system. In the presence of thiamine pyrophosphate, pyruvate, coenzyme A, and Mg2+, E3 inactivation by BrCH2CONHPhAsO was selective (coenzyme A binding was unaffected) and stoichiometrically related to PD complex inactivation, indicating that a complement of E3 is necessary for full complex activity. The activity of the pyruvate dehydrogenase (E1) component was unaltered by BrCH2-CONHPhAsO in both systems. On inhibition of the PD complex with BrCH2CONHPhAsO, the reagent mediated interchain cross-linking between E2 and about half of the E3 subunits. A marked change occurred in the quaternary structure of the PD complex, with some E1 and E3 subunits being dissociated from the E2 core. The mechanism outlined by Stevenson et al. [Stevenson, K. J., Hale, G., & Perham, R. N. (1978) Biochemistry 17, 2189] for the inhibition of the PD complex by BrCH2CONHPhAsO must be revised on the basis of these findings. E3 is only partially modified by delivery of the bromoacetyl moiety of the bifunctional reagent (covalently attached to lipoyl residues of E2 through dithioarsinite bonds) into the active site of bound E3. The inhibition of E3, dissociated from the PD complex during cross-linking, likely occurs via direct interaction of the free enzyme with BrCH2CONHPhAsO by initial dithioarsinite modification of the reduced active-site disulfide followed by alkylation of a nearby residue.

Arsenicals↗
PubMed1997

Mutations in PDX1, the human lipoyl-containing component X of the pyruvate dehydrogenase-complex gene on chromosome 11p1, in congenital lactic acidosis.

We have identified and sequenced a cDNA that encodes an apparent human orthologue of a yeast protein-X component (ScPDX1) of pyruvate dehydrogenase multienzyme complexes. The new human cDNA that has been referred to as "HsPDX1" cDNA was cloned by use of the "database cloning" strategy and had a 1,506-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 20% identical with that encoded by the yeast PDX1 gene and 40% identical with that encoded by the lipoate acetyltransferase component of the pyruvate dehydrogenase and included a lipoyl-bearing domain that is conserved in some dehydrogenase enzyme complexes. Northern blot analysis demonstrated that the major HsPDX1 mRNA was 2.5 kb in length and was expressed mainly in human skeletal and cardiac muscles but was also present, at low levels, in other tissues. FISH analysis performed with a P1-derived artificial chromosome (PAC)-containing HsPDX1 gene sublocalized the gene to 11p1.3. Molecular investigation of PDX1 deficiency in four patients with neonatal lactic acidemias revealed mutations 78del85 and 965del59 in a homozygous state, and one other patient had no PDX1 mRNA expression.

Acidosis, Lactic↗
PubMed1990

Application of high-performance liquid chromatography to the purification, disintegration and molecular mass determination of pyruvate dehydrogenase multi-enzyme complexes from different sources.

The pyruvate dehydrogenase complex is associated with the inner mitochondrial membrane. A gentle and rapid purification procedure, especially for the very unstable pyruvate dehydrogenase complex from the extremely thermophilic organism Thermus aquaticus, is described. This procedure is based essentially on a combination of hydrophobic interaction and of adsorption chromatography by the rapid fast protein liquid chromatographic technique. Applying the same method, a relative molecular mass of 9.1 . 10(6) daltons was obtained by gel filtration on Superose 6 HR 10/30 for the pyruvate dehydrogenase complex from T. aquaticus. The same column served to resolve the pyruvate dehydrogenase complex into its enzyme components.

Animals↗
PubMed1989

Antibodies against an inter-domain segment of polypeptide chain inhibit active-site coupling in the pyruvate dehydrogenase multienzyme complex.

A synthetic peptide, AAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKA, proved to be an efficient and specific immunogen in rabbits. The amino acid sequence of the peptide is identical to that of the inter-domain region (PEP3) linking the innermost of the three lipoyl domains to the dihydrolipoamide dehydrogenase-binding domain in the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli. Fab fragments from anti-PEP3 antibodies selectively inhibited active-site coupling in the complex without affecting the individual activities of the three component enzymes, highlighting the role of the inter-domain regions as flexible linkers in catalysis.

Antibodies↗
PubMed1986

Pyruvate dehydrogenase and 3-fluoropyruvate: chemical competence of 2-acetylthiamin pyrophosphate as an acetyl group donor to dihydrolipoamide.

The pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex catalyzes the decomposition of 3-fluoropyruvate to CO2, fluoride anion, and acetate. Acetylthiamin pyrophosphate (acetyl-TPP) is an intermediate in this reaction. Incubation of the pyruvate dehydrogenase complex with 3-fluoro[1,2-14C]pyruvate, TPP, coenzyme A (CoASH), and either NADH or pyruvate as reducing systems leads to the formation of [14C]acetyl-CoA. In this reaction the acetyl group of acetyl-TPP is partitioned by transfer to both CoASH (87 +/- 2%) and water (13 +/- 2%). When the E1 component is incubated with 3-fluoro[1,2-14C]pyruvate, TPP, and dihydrolipoamide, [14C]acetyldihydrolipoamide is produced. The formation of [14C]acetyldihydrolipoamide was examined as a function of dihydrolipoamide concentration (0.25-16 mM). A plot of the extent of acetyl group partitioning to dihydrolipoamide as a function of 1/[dihydrolipoamide] showed 95 +/- 2% acetyl group transfer to dihydrolipoamide when dihydrolipoamide concentration was extrapolated to infinity. It is concluded that acetyl-TPP is chemically competent as an intermediate for the pyruvate dehydrogenase complex catalyzed oxidative decarboxylation of pyruvate.

Carbon Radioisotopes↗
PubMed1983

Stereoisomers of tetrahydrothiamin pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

Tetrahydrothiamin pyrophosphate, an analogue of thiamin pyrophosphate (TPP) in which the thiazolium ring has been reduced to a thiazolidine ring, was prepared by borohydride reduction of TPP. It consists of four stereoisomers, comprising two diastereomers each of which is a racemic mixture, generated by the introduction of two chiral centers on the thiazolidine ring. The major and minor diastereomers were separated and inferred to be of the cis and trans configurations, respectively, from a study of the nuclear Overhauser effects in the 1H NMR spectrum of the simpler tetrahydrothiamin. Tetrahydro-TPP behaves as a mixture of potent inhibitors of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex from Escherichia coli. The site of binding is probably the TPP-binding site on E1, and the Kd for each of the four stereoisomers was estimated. The cis isomers of tetrahydro-TPP bind more tightly than does TPP, whereas the trans isomers appear to bind with about the same Kd as TPP. Sodium borohydride caused a rapid inhibition of E1 activity in the presence of TPP, believed to be due to reaction of borohydride with enzyme-bound TPP. The experiments are consistent with an enhancement of the reactivity of the thiazole ring of TPP when bound to the catalytic site of E1, which could be due to polarization of the greater than +N=C bond near a hydrophobic or positively charged region of the protein. A spontaneous reactivation occurred after the initial inhibition by borohydride, attributable to a weakly binding inhibitor, not tetrahydro-TPP, being formed at the catalytic site.

Borohydrides↗
PubMed2004

R-lipoic acid inhibits mammalian pyruvate dehydrogenase kinase.

The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects.

Acetyltransferases↗
PubMed1991

Specific reactivity of recombinant human PDC-E1 alpha in primary biliary cirrhosis.

The mitochondrial autoantigens recognized by autoantibodies in patients with primary biliary cirrhosis have been identified as components of related multi-enzyme complexes, including acyltransferases of the pyruvate dehydrogenase complex (PDC), the branched-chain alpha-keto acid dehydrogenase complex (BCODH), the alpha-ketoglutarate dehydrogenase complex (OGDC), protein X and pyruvate dehydrogenase (PDC) E1 alpha and E1 beta. The major autoantigens, PDC-E2, BCODH-E2 and OGDC-E2, share some sequence homology; the epitopes on these antigens appear to be close to, or identical with, the lipoic acid binding site. Furthermore, all three antigens share some structural homology. In contrast, antibodies to PDC-E1 alpha are present in lower titers, and have been more difficult to detect. PDC-E1 alpha also differs from the three major autoantigens in that it lacks any covalently bound lipoic acid. PDC-E1 alpha cannot be purified in large quantities and becomes unstable in the absence of PDC-E1 beta. To address these problems, we have subcloned recombinant human PDC-E1 alpha to pGEX, pGEX is a vector which produces a recombinant polypeptide fused to glutathione S-transferase. The resultant E1 alpha fusion protein is stable and has a low background in immunoassays. Using the recombinant protein, we have developed an ELISA that allows rapid and reproducible quantification of antibodies to human PDC-E1 alpha. Finally, we demonstrate that a major epitope on PDC-E1 alpha is within a 300 amino acid region that contains the enzyme functional sites, namely the phosphorylation site and the TPP binding site.

Autoantibodies↗
PubMed1982

Role of pyruvate transporter in the regulation of the pyruvate dehydrogenase multienzyme complex in perfused rat liver.

Metabolic substrates such as octanoate, beta-hydroxybutyrate, and alpha-ketoisocaproate which produce acetoacetate stimulate the rate of pyruvate decarboxylation in perfused livers from fed rats at perfusate pyruvate concentrations in the physiological range (below 0.2 mM). A quantitative relationship between pyruvate oxidation (14CO2 production from [1-14C]pyruvate) and ketogenesis (production of acetoacetate or total ketone bodies) was observed with all ketogenic substrates when studied over a wide range of concentrations. The ratio of extra pyruvate decarboxylated to extra acetoacetate produced was greater than 1 with octanoate and alpha-ketoisocaproate, but it was less than 1 with beta-hydroxybutyrate. The stimulatory effect of beta-hydroxybutyrate on pyruvate decarboxylation was abolished completely in the presence of 0.1 mM alpha-cyanocinnamate, an inhibitor of the pyruvate transporting system in the mitochondrial membrane. The data suggest that the mechanism by which the flux through the pyruvate dehydrogenase reaction is stimulated in liver under ketogenic conditions involves an acceleration of the net rate of pyruvate transport into the mitochondria compartment due to an exchange with acetoacetate and/or acetoacetate plus beta-hydroxybutyrate.

Acetoacetates↗
PubMed2006

Active-site changes in the pyruvate dehydrogenase multienzyme complex E1 apoenzyme component from Escherichia coli observed at 2.32 A resolution.

The first enzymatic component, E1 (EC 1.2.4.1), of the pyruvate dehydrogenase multienzyme complex (PDHc) utilizes thiamine diphosphate (ThDP) and Mg(2+) as cofactors. The structure of a branched-chain-specific E1 apoenzyme from the heterotetrameric alpha(2)beta(2) E1 family was recently reported and showed that disorder-to-order transformations in two active-site loops take place upon cofactor binding. To ascertain what effect the absence of cofactor may have in the homodimeric alpha(2) Escherichia coli PDHc E1, the corresponding apoenzyme has been prepared and its three-dimensional structure determined and analyzed at 2.32 A by crystallographic methods. This represents the first reported apoenzyme structure for any E1 component from the homodimeric alpha(2) family. Electron-density features occurring in the region where the cofactor pyrimidine ring would normally be expected to bind are of size, shape and location compatible with water molecules that form a hydrogen-bonded linkage between residues Glu571 and Val192, which normally make conserved interactions with the ThDP cofactor. A histidine side chain that normally forms hydrogen bonds to ThDP is disordered in its absence and partially occupies two sites. Unlike in the reported heterotetrameric branched-chain apo-E1, no disorder/order loop transformations are evident in apo-PDHc E1 relative to the holo-E1 enzyme (PDHc E1-ThDP-Mg(2+)). Differences in the extent of hydrogen-bonding networks found in the apo-E1 enzyme, the holo-E1 enzyme and in an inhibitor complex with bound thiamine 2-thiazolone diphosphate (ThTDP), PDHc E1-ThTDP-Mg(2+), are described.

Apoenzymes↗
PubMed1975

Absence of pyruvate decarboxylase activity in man: a cause of congenital lactic acidosis.

A complete deficiency in the pyruvate dehydrogenase system activity contributed to the death of a 6-month-old infant with congenital lactic acidosis. The enzymatic block could be isolated to the first component, pyruvate decarboxylase (E1) of the pyruvate dehydrogenase complex. This enzymatic deficiency allowed a demonstration of an "intercomplex" exchange of the components of the mammalian pyruvate dehydrogenase system and indicated that the first component is normally present in an apparent excess.

Acidosis↗
PubMed1997

2-Oxo-3-alkynoic acids, universal mechanism-based inactivators of thiamin diphosphate-dependent decarboxylases: synthesis and evidence for potent inactivation of the pyruvate dehydrogenase multienzyme complex.

A new class of compounds, the 2-oxo-3-alkynoic acids with a phenyl substituent at carbon 4 was reported by the authors as potent irreversible and mechanism-based inhibitors of the thiamin diphosphate- (ThDP-) dependent enzyme pyruvate decarboxylase [Chiu, C.-F., & Jordan, F. (1994) J. Org. Chem. 59, 5763-5766]. The method has been successfully extended to the synthesis of the 4-, 5-, and 7-carbon aliphatic members of this family of compounds. These three compounds were then tested on three ThDP-dependent pyruvate decarboxylases: the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) and its E1 (ThDP-dependent) component, pyruvate oxidase (POX, phosphorylating; from Lactobacillus plantarum),and pyruvate decarboxylase (PDC) from Saccharomycescerevisiae. All three enzymes were irreversibly inhibited by the new compounds. The 4-carbon acid is the best substrate-analog inactivator known to date for PDHc, more potent than either fluoropyruvate or bromopyruvate. The following conclusions were drawn from extensive studies with PDHc: (a) The kinetics of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is time- and concentration-dependent. (b) The 4-carbon acid has a Ki 2 orders of magnitude stronger than the 5-carbon acid, clearly demonstrating the substrate specificity of PDHc. (c) The rate of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is enhanced by the addition of ThDP and MgCl2. (d) Pyruvate completely protects E1 and partially protects PDHc from inactivation by 2-oxo-3-butynoic acid. (e) E1 but not E2-E3 is the target of inactivation by 2-oxo-3-butynoic acid. (f) Inactivation of E1 by 2-oxo-3-butynoic acid is accompanied by modification of 1.3 cysteines/E1 monomer. The order of reactivity with the 4-carbon acid was PDHc > POX > PDC. While the order of reactivity with PDHc and POX was 2-oxo-3-butynoic acid > 2-oxo-3-pentynoic acid > 2-oxo-3-heptynoic acid, the order of reactivity was reversed with PDC.

Bacterial Proteins↗
PubMed1992

Interaction of avidin with the lipoyl domains in the pyruvate dehydrogenase multienzyme complex: three-dimensional location and similarity to biotinyl domains in carboxylases.

Avidin can form intermolecular cross-links between particles of the pyruvate dehydrogenase multienzyme complex from various sources. Avidin does this by binding to lipoic acid-containing regions of the dihydrolipoamide acetyltransferase polypeptide chains that comprise the structural core of the complex. It is inferred that the lipoyl domains of the acetyltransferase chain extend outwards from the interior of the enzyme particle, interdigitating between the subunits of the other two enzymes bound peripherally in the assembled structure, with the lipoyl-lysine residues capable of reaching to within at least 1-2 nm of the outer surface of the enzyme complex (diameter ca. 37 nm). The distribution of enzymic activities between different domains of the dihydrolipoamide acetyltransferase chain implies that considerable movement of the lipoyl domains is a feature of the catalytic activity of the enzyme complex. There is evidence that the lipoyl domain of the 2-oxo acid dehydrogenase complexes is similar in structure to a domain that binds the cofactor biotin, also in amide linkage with a specific lysine residue, in the biotin-dependent class of carboxylases.

Amidohydrolases↗
PubMed1997

Metabolic responses of pyruvate decarboxylase-negative Saccharomyces cerevisiae to glucose excess.

In Saccharomyces cerevisiae, oxidation of pyruvate to acetyl coenzyme A can occur via two routes. In pyruvate decarboxylase-negative (Pdc-) mutants, the pyruvate dehydrogenase complex is the sole functional link between glycolysis and the tricarboxylic acid (TCA) cycle. Such mutants therefore provide a useful experimental system with which to study regulation of the pyruvate dehydrogenase complex. In this study, a possible in vivo inactivation of the pyruvate dehydrogenase complex was investigated. When respiring, carbon-limited chemostat cultures of wild-type S. cerevisiae were pulsed with excess glucose, an immediate onset of respiro-fermentative metabolism occurred, accompanied by a strong increase of the glycolytic flux. When the same experiment was performed with an isogenic Pdc- mutant, only a small increase of the glycolytic flux was observed and pyruvate was the only major metabolite excreted. This finding supports the hypothesis that reoxidation of cytosolic NADH via pyruvate decarboxylase and alcohol dehydrogenase is a prerequisite for high glycolytic fluxes in S. cerevisiae. In Pdc- cultures, the specific rate of oxygen consumption increased by ca. 40% after a glucose pulse. Calculations showed that pyruvate excretion by the mutant was not due to a decrease of the pyruvate flux into the TCA cycle. We therefore conclude that rapid inactivation of the pyruvate dehydrogenase complex (e.g., by phosphorylation of its E1 alpha subunit, a mechanism demonstrated in many higher organisms) is not a relevant mechanism in the response of respiring S. cerevisiae cells to excess glucose. Consistently, pyruvate dehydrogenase activities in cell extracts did not exhibit a strong decrease after a glucose pulse.

Acetyl Coenzyme A↗
PubMed2003

Induction of specific T-helper and cytolytic responses to epitopes displayed on a virus-like protein scaffold derived from the pyruvate dehydrogenase multienzyme complex.

The icosahedral protein scaffold (1.5MDa) generated by self-assembly of the catalytic domains of the dihydrolipoyl acetyltransferase core of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus has been engineered to display 60 copies of one or more peptide epitopes on a single molecule (E2DISP). An E2DISP scaffold displaying pep23, a 15-residue B- and T-helper epitope from the reverse transcriptase of HIV-1, was able to induce a pep23-specific T-helper response in cell lines in vitro. The same scaffold displaying both pep23 and peptide RT2, a nine-residue CTL epitope from HIV-1 reverse transcriptase, was able to prime an RT2-specific CD8(+) T-cell response in human cell lines in vitro and in HLA-A2 transgenic mice in vivo. This was accompanied by a humoral antibody response specific for E2DISP-presented epitopes. Thus, the icosahedral acetyltransferase core constitutes a simple and flexible scaffold for multiple epitope display with access to both cellular and humoral immune response pathways.

Acetyltransferases↗
PubMed1998

Arginine 302 mutations in the pyruvate dehydrogenase E1alpha subunit gene: identification of further patients and in vitro demonstration of pathogenicity.

Three further patients with mutations in the codon for arginine 302 of the E1alpha subunit of the pyruvate dehydrogenase complex have been identified. Mutations in this codon have now been found in nine patients with pyruvate dehydrogenase deficiency in seven unrelated families, in sharp contrast to the great majority of other PDH E1alpha mutations which have been described in single individuals only. Because of the relatively high frequency of this mutation and because very few PDH E1alpha mutations have been demonstrated to be causative, we have established a system for analysing the consequences of defined mutations using transfection of normal and mutant PDH E1alpha cDNA into transformed human fibroblasts which have no endogenous E1alpha mRNA or protein. Using this test system, we have demonstrated that the R302C mutation results in the production of PDH E1alpha protein which is devoid of enzymic activity.

Arginine↗