Showing papers on "Oxoglutarate dehydrogenase complex published in 2001"

Beta-amyloid inhibits integrated mitochondrial respiration and key enzyme activities.

Abstract: Disrupted energy metabolism, in particular reduced activity of cytochrome oxidase (EC 1.9.3.1), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) and pyruvate dehydrogenase (EC 1.2.4.1) have been reported in post-mortem Alzheimer's disease brain. beta-Amyloid is strongly implicated in Alzheimer's pathology and can be formed intracellularly in neurones. We have investigated the possibility that beta-amyloid itself disrupts mitochondrial function. Isolated rat brain mitochondria have been incubated with the beta-amyloid alone or together with nitric oxide, which is known to be elevated in Alzheimer's brain. Mitochondrial respiration, electron transport chain complex activities, alpha-ketoglutarate dehydrogenase activity and pyruvate dehydrogenase activity have been measured. Beta-amyloid caused a significant reduction in state 3 and state 4 mitochondrial respiration that was further diminished by the addition of nitric oxide. Cytochrome oxidase, alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase activities were inhibited by beta-amyloid. The K(m) of cytochrome oxidase for reduced cytochrome c was raised by beta-amyloid. We conclude that beta-amyloid can directly disrupt mitochondrial function, inhibits key enzymes and may contribute to the deficiency of energy metabolism seen in Alzheimer's disease.

Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites.

Abstract: The enzymic activity of the mammalian pyruvate dehydrogenase complex is regulated by the phosphorylation of three serine residues (sites 1, 2 and 3) located on the E1 component of the complex. Here we report that the four isoenzymes of protein kinase responsible for the phosphorylation and inactivation of pyruvate dehydrogenase (PDK1, PDK2, PDK3 and PDK4) differ in their abilities to phosphorylate the enzyme. PDK1 can phosphorylate all three sites, whereas PDK2, PDK3 and PDK4 each phosphorylate only site 1 and site 2. Although PDK2 phosphorylates site 1 and 2, it incorporates less phosphate in site 2 than PDK3 or PDK4. As a result, the amount of phosphate incorporated by each isoenzyme decreases in the order PDK1>PDK3>or=PDK4>PDK2. Significantly, binding of the coenzyme thiamin pyrophosphate to pyruvate dehydrogenase alters the rates and stoichiometries of phosphorylation of the individual sites. First, the rate of phosphorylation of site 1 by all isoenzymes of kinase is decreased. Secondly, thiamin pyrophosphate markedly decreases the amount of phosphate that PDK1 incorporates in sites 2 and 3 and that PDK2 incorporates in site 2. In contrast, the coenzyme does not significantly affect the total amount of phosphate incorporated in site 2 by PDK3 and PDK4, but instead decreases the rate of phosphorylation of this site. Furthermore, pyruvate dehydrogenase complex phosphorylated by the individual isoenzymes of kinase is reactivated at different rates by pyruvate dehydrogenase phosphatase. Both isoenzymes of phosphatase (PDP1 and PDP2) readily reactivate the complex phosphorylated by PDK2. When pyruvate dehydrogenase is phosphorylated by other isoenzymes, the rates of reactivation decrease in the order PDK4>or=PDK3>PDK1. Taken together, results reported here strongly suggest that the major determinants of the activity state of pyruvate dehydrogenase in mammalian tissues include the phosphorylation site specificity of isoenzymes of kinase in addition to the absolute amounts of kinase and phosphatase protein expressed in mitochondria.

Yeast mitochondrial dehydrogenases are associated in a supramolecular complex.

Abstract: Separation of yeast mitochondrial complexes by colorless native polyacrylamide gel electrophoresis led to the identification of a supramolecular structure exhibiting NADH−dehydrogenase activity. Components of this complex were identified by N-terminal Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. The complex was found to contain the five known intermembrane space-facing dehydrogenases, namely two external NADH−dehydrogenases Nde1p and Nde2p, glycerol-3-phosphate dehydrogenase Gut2p, d- and l-lactate-dehydrogenases Dld1p and Cyb2p, the matrix-facing NADH−dehydrogenase Ndi1p, two probable flavoproteins YOR356Wp and YPR004Cp, four tricarboxylic acids cycle enzymes (malate dehydrogenase Mdh1p, citrate synthase Cit1p, succinate dehydrogenase Sdh1p, and fumarate hydratase Fum1p), and the acetaldehyde dehydrogenase Ald4p. The association of these proteins is discussed in terms of NADH-channeling.

Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae.

Abstract: This work was undertaken to clarify the role of acetaldehydedehydrogenases in Saccharomyces cerevisiae metabolismduring growth on respiratory substrates. Until now, there hasbeen little agreement concerning the ability of mutantsdeleted in gene ALD4, encoding mitochondrial acetaldehydedehydrogenase, to grow on ethanol. Therefore we con-structed mutants in two parental strains (YPH499 andW303-1a). Some differences appeared in the growthcharacteristics of mutants obtained from these two parentalstrains. For these experiments we used ethanol, pyruvate orlactate as substrates. Mitochondria can oxidize lactate intopyruvate using an ATP synthesis-coupled pathway. Theald4Dmutant derived from the YPH499 strain failed togrow on ethanol, but growth was possible for the ald4Dmutant derived from the W303-1a strain. The co-disruptionof ALD4 and PDA1 (encoding subunit E1a of pyruvatedehydrogenase) prevented the growth on pyruvate for bothstrains but prevented growth on lactate only in the doublemutant derived from the YPH499 strain, indicating that themutation effects are strain-dependent. To understand thesedifferences, we measured the enzyme content of thesedifferent strains. We found the following: (a) the activity ofcytosolic acetaldehyde dehydrogenase in YPH499 wasrelatively low compared to the W303-1a strain; (b) it waspossible to restore the growth of the mutant derived fromYPH499 either by addition of acetate in the media or byintroduction into this mutant of a multicopy plasmidcarrying the ALD6 gene encoding cytosolic acetaldehydedehydrogenase. Therefore, the lack of growth of the mutantderived from the YPH499 strain seemed to be related to thelow activity of acetaldehyde oxidation. Therefore, whencultured on ethanol, the cytosolic acetaldehyde dehydro-genase can partially compensate for the lack of mitochon-drial acetaldehyde dehydrogenase only when the activity ofthe cytosolic enzyme is sufficient. However, when culturedon pyruvate and in the absence of pyruvate dehydrogenase,the cytosolic acetaldehyde dehydrogenase cannot compen-sate for the lack of the mitochondrial enzyme because themitochondrial form produces intramitochondrial NADH andconsequently ATP through oxidative phosphorylation.Keywords: Saccharomycescerevisiae; acetaldehydedehydro-genase; pyruvate dehydrogenase.This work was undertaken to clarify the role of acetaldehydedehydrogenases in Saccharomyces cerevisiae metabolismduring growth on respiratory substrates. Three conversionpathways of pyruvate into acetyl-CoA have been describedin yeast (Fig. 1). The pyruvate dehydrogenase complexlocated inside the mitochondrial matrix converts pyruvateinto acetyl-CoA with the production of NADH. This com-plex has been purified from S. cerevisiae [1,2] and its expres-sion is independent of the carbon source used for growth [3].Another metabolic pathway occurs via the cytosolic pyru-vate dehydrogenase bypass [4] and requires the followingenzymes: pyruvate decarboxylase, cytosolic acetaldehydedehydrogenase and acetyl-CoA synthase. All these enzymesare located in the cytosol. This appears to be an alternativepathway for the production of cytoplasmic acetyl-CoA forbiosynthesis. Recently, a mitochondrial pyruvate dehydro-genase bypass was described, showing that pyruvate can beoxidized inside mitochondria by a pathway involving mito-chondrial acetaldehyde dehydrogenase [5]. Pyruvate is firstdecarboxylated to acetaldehyde in the cytosol by pyruvatedecarboxylase and is then oxidized by mitochondrial acet-aldehyde dehydrogenase, leading to the reduction of NAD

A Novel Syndrome Affecting Multiple Mitochondrial Functions, Located by Microcell-Mediated Transfer to Chromosome 2p14-2p13

Abstract: We have studied cultured skin fibroblasts from three siblings and one unrelated individual, all of whom had fatal mitochondrial disease manifesting soon after birth. After incubation with 1 mM glucose, these four cell strains exhibited lactate/pyruvate ratios that were six times greater than those of controls. On further analysis, enzymatic activities of the pyruvate dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, NADH cytochrome c reductase, succinate dehydrogenase, and succinate cytochrome c reductase were severely deficient. In two of the siblings the enzymatic activity of cytochrome oxidase was mildly decreased (by ∼50%). Metabolite analysis performed on urine samples taken from these patients revealed high levels of glycine, leucine, valine, and isoleucine, indicating abnormalities of both the glycine-cleavage system and branched-chain α-ketoacid dehydrogenase. In contrast, the activities of fibroblast pyruvate carboxylase, mitochondrial aconitase, and citrate synthase were normal. Immunoblot analysis of selected complex III subunits (core 1, cyt c 1 , and iron-sulfur protein) and of the pyruvate dehydrogenase complex subunits revealed no visible changes in the levels of all examined proteins, decreasing the possibility that an import and/or assembly factor is involved. To elucidate the underlying molecular defect, analysis of microcell-mediated chromosome-fusion was performed between the present study's fibroblasts (recipients) and a panel of A9 mouse:human hybrids (donors) developed by Cuthbert et al. (1995). Complementation was observed between the recipient cells from both families and the mouse:human hybrid clone carrying human chromosome 2. These results indicate that the underlying defect in our patients is under the control of a nuclear gene, the locus of which is on chromosome 2. A 5-cM interval has been identified as potentially containing the critical region for the unknown gene. This interval maps to region 2p14-2p13.

Regulation of Branched-Chain α-Keto Acid Dehydrogenase Kinase Expression in Rat Liver

Abstract: Branched-chain amino acids are toxic in excess but have to be conserved for protein synthesis. This is accomplished in large part by control of the activity of the branched-chain alpha-keto acid dehydrogenase complex by phosphorylation/dephosphorylation. Regulation of the activity of the hepatic enzyme appears particularly important, at least in rats, since an exceptional high activity of the complex in this tissue makes the liver the primary clearing house for excess branched-chain alpha-keto acids released by other tissues. The degree to which the branched-chain alpha-keto acid dehydrogenase complex is inactivated by phosphorylation is determined by the activity of the branched-chain alpha-keto acid dehydrogenase kinase, which is itself regulated by allosteric effectors as well as factors that affect its level of expression. Well established among these are the alpha-keto acid produced by leucine transamination, which is a potent inhibitor of the kinase, and starvation for dietary protein, which causes increased expression of the branched-chain alpha-keto acid dehydrogenase kinase. The latter finding resulted in the working hypothesis that nutrients and hormones regulate expression of the branched-chain alpha-keto acid dehydrogenase kinase. Evidence has been obtained for the involvement of thyroid hormone, glucocorticoids and ligands for peroxisome proliferator-activated receptor alpha. Thyroid hormone induces, whereas glucocorticoids and peroxisome proliferator-activated receptor alpha ligands repress, expression of the kinase. Increased blood levels of thyroid hormone are proposed to be responsible for increased expression of branched-chain alpha-keto acid dehydrogenase kinase in animals starved for protein.

Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms.

Abstract: The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.

The mitochondrial oxoglutarate carrier: cysteine-scanning mutagenesis of transmembrane domain IV and sensitivity of Cys mutants to sulfhydryl reagents.

Abstract: Using a functional mitochondrial oxoglutarate carrier mutant devoid of Cys residues (C-less carrier), each amino acid residue in transmembrane domain IV and flanking hydrophilic loops (from T179 to S205) was replaced individually with Cys. The great majority of the 27 mutants exhibited significant oxoglutarate transport in reconstituted liposomes as compared to the activity of the C-less carrier. In contrast, Cys substitution for G183, R190, Q198, and Y202, in either C-less or wild-type carriers, yielded molecules with complete loss of oxoglutarate transport activity. G183 and R190 could be partially replaced only by Ala and Lys, respectively, whereas Q198 and Y202 were irreplaceable with respect to oxoglutarate transport. Of the single-Cys mutants tested, only T187C, A191C, V194C, and N195C were strongly inactivated by N-ethylmaleimide and by low concentrations of methanethiosulfonate derivatives. Oxoglutarate protects Cys residues at positions 187, 191, and 194 against reaction with N-ethylmaleimide. These positions as well as the residues found to be essential for the carrier activity, except Y202 which is located in the extramembrane loop IV-V, reside on the same face of transmembrane helix IV, probably lining part of a water-accessible crevice or channel between helices of the oxoglutarate carrier.

Binding, hydration, and decarboxylation of the reaction intermediate glutaconyl-coenzyme A by human glutaryl-CoA dehydrogenase.

Abstract: Glutaconyl-coenzyme A (CoA) is the presumed enzyme-bound intermediate in the oxidative decarboxylation of glutaryl-CoA that is catalyzed by glutaryl-CoA dehydrogenase. We demonstrated glutaconyl-CoA bound to glutaryl-CoA dehydrogenase after anaerobic reduction of the dehydrogenase with glutaryl-CoA. Glutaryl-CoA dehydrogenase also has intrinsic enoyl-CoA hydratase activity, a property of other members of the acyl-CoA dehydrogenase family. The enzyme rapidly hydrates glutaconyl-CoA at pH 7.6 with a kcat of 2.7 s-1. The kcat in the overall oxidation−decarboxylation reaction at pH 7.6 is about 9 s-1. The binding of glutaconyl-CoA was quantitatively assessed from the Km in the hydratase reaction, 3 μM, and the Ki, 1.0 μM, as a competitive inhibitor of the dehydrogenase. These values compare with Km and Ki of 4.0 and 12.9 μM, respectively, for crotonyl-CoA. Glu370 is the general base catalyst in the dehydrogenase that abstracts an α-proton of the substrate to initiate the catalytic pathway. The mutant dehydrog...

The A245K mutation exposes another stage of the bacterial L-lactate dehydrogenase reaction mechanism.

Abstract: The A245K mutant of Bacillus stearothermophilus l-lactate dehydrogenase has been expressed in Escherichia coli and purified. A qualitative change in the reaction mechanism prior to the hydride transfer step in the reverse direction in the mutant is revealed. Both transient and steady state characteristics of the mutant are presented and show in contrast to the wild-type enzyme where a rearrangement of an enzyme−NADH−pyruvate complex is rate-limiting that in the mutant the rearrangement is much faster and hydride transfer is the first slow step. The steady state is limited by a new second slower conformation change involving an NAD+ complex. The mutation may provide a valuable framework for inhibitor and drug design research.

Pyruvate dehydrogenase from Azotobacter vinelandii. Properties of the N-terminally truncated enzyme.

Abstract: The pyruvate dehydrogenase multienzyme complex (PDHC) catalyses the oxidative decarboxylation of pyruvate and the subsequent acetylation of coenzyme A to acetyl-CoA. Previously, limited proteolysis experiments indicated that the N-terminal region of the homodimeric pyruvate dehydrogenase (E1p) from Azotobacter vinelandii could be involved in the binding of E1p to the core protein (E2p) [Hengeveld, A. F., Westphal, A. H. & de Kok, A. (1997) Eur J. Biochem. 250, 260-268]. To further investigate this hypothesis N-terminal deletion mutants of the E1p component of Azotobacter vinelandii pyruvate dehydrogenase complex were constructed and characterized. Up to nine N-terminal amino acids could be removed from E1p without effecting the properties of the enzyme. Truncation of up to 48 amino acids did not effect the expression or folding abilities of the enzyme, but the truncated enzymes could no longer interact with E2p. The 48 amino acid deletion mutant (E1pdelta48) is catalytically fully functional: it has a Vmax value identical to that of wild-type E1p, it can reductively acetylate the lipoamide group attached to the lipoyl domain of the core enzyme (E2p) and it forms a dimeric molecule. In contrast, the S0.5 for pyruvate is decreased. A heterodimer was constructed containing one subunit of wild-type E1p and one subunit of E1pdelta48. From the observation that the heterodimer was not able to bind to E2p, it is concluded that both N-terminal domains are needed for the binding of E1p to E2p. The interactions are thought to be mainly of an electrostatic nature involving negatively charged residues on the N-terminal domains of E1p and previously identified positively charged residues on the binding and catalytic domain of E2p.

Improvement of cell culture performance by inhibitors of pyruvate dehydrogenase kinase

Abstract: The invention provides methods and compositions for improved in vitro culture of animal cells that make use of inhibitors of pyruvate dehydrogenase kinase. Particularly preferred inhibitors are salts of dichloroacetic acid such as sodium dichloroacetate.

Interaction of 3,4-dienoyl-CoA thioesters with medium chain acyl-CoA dehydrogenase: stereochemistry of inactivation of a flavoenzyme.

Abstract: The medium chain acyl-CoA dehydrogenase is rapidly inhibited by racemic 3,4-dienoyl-CoA derivatives with a stoichiometry of two molecules of racemate per enzyme flavin. Synthesis of R- and S-3,4-decadienoyl-CoA shows that the R-enantiomer is a potent, stoichiometric, inhibitor of the enzyme. alpha-Proton abstraction yields an enolate to oxidized flavin charge-transfer intermediate prior to adduct formation. The crystal structure of the reduced, inactive enzyme shows a single covalent bond linking the C-4 carbon of the 2,4-dienoyl-CoA moiety and the N5 locus of reduced flavin. The kinetics of reversal of adduct formation by release of the conjugated 2,4-diene were evaluated as a function of both acyl chain length and truncation of the CoA moiety. The adduct is most stable with medium chain length allenic inhibitors. However, the adducts with R-3,4-decadienoyl-pantetheine and -N-acetylcysteamine are some 9- and >100-fold more kinetically stable than the full-length CoA thioester. Crystal structures of these reduced enzyme species, determined to 2.4 A, suggest that the placement of H-bonds to the inhibitor carbonyl oxygen and the positioning of the catalytic base are important determinants of adduct stability. The S-3,4-decadienoyl-CoA is not a significant inhibitor of the medium chain dehydrogenase and does not form a detectable flavin adduct. However, the S-isomer is rapidly isomerized to the trans-trans-2,4-conjugated diene. Protein modeling studies suggest that the S-enantiomer cannot approach close enough to the isoalloxazine ring to form a flavin adduct, but can be facilely reprotonated by the catalytic base. These studies show that truncation of CoA thioesters may allow the design of unexpectedly potent lipophilic inhibitors of fatty acid oxidation.

2-Oxoglutarate transport system in Staphylococcus aureus.

Abstract: 2-[14C]oxoglutarate uptake in resting cells of Staphylococcus aureus 17810S occurs via two kinetically different systems: (1) a secondary, electrogenic 2-oxoglutarate:H+ symporter (Km=0.105 mM), energized by an electrochemical proton potential (ΔµH+) that is generated by the oxidation of endogenous amino acids and sensitive to ionophores, and (2) a ΔµH+-independent facilitated diffusion system (Km=1.31 mM). The 2-oxoglutarate transport system of S. aureus 17810S can be classified as a new member of the MHS (metabolite:H+ symporter) family. This transporter takes up various dicarboxylic acids in the order of affinity: succinate = malate > fumarate > 2-oxoglutarate > glutamate. Energy conservation with 2-oxoglutarate was studied in starved cells of strain 17810S. Initial transport of 2-oxoglutarate in these cells is energized by ΔµH+ generated via hydrolysis of residual ATP. Subsequent oxidation of the accumulated 2-oxoglutarate generates ΔµH+ for further, autoenergized transport of this 2-oxoacid and also for ΔµH+-linked resynthesis of ATP. In the cadmium-sensitive S. aureus 17810S, Cd2+ accumulation strongly inhibits energy conservation with 2-oxoglutarate at the level of ΔµH+ generation, without direct blocking of the 2-oxoglutarate transport system or ATP synthase complex. In the cadmium-resistant S. aureus 17810R, Cd2+ does not affect energy conservation due to its extrusion by the Cd2+ efflux system (Cd2+-ATPase of P-type), which prevents Cd2+ accumulation.

Metabolic transitions and the role of the pyruvate dehydrogenase complex during development of Ascaris suum.

Abstract: The parasitic nematode, Ascaris suum, undergoes a number of wellcharacterized metabolic transitions during its development (Table 14.1), but little is known about the regulation of these events (Barrett, 1976; Komuniecki and Komuniecki, 1995). Adults reside in the porcine small intestine and fertilization takes place under low oxygen tensions. The unembryonated ‘egg’ that leaves the host is metabolically quiescent, has no detectable cytochrome oxidase activity or ubiquinone and appears to be transcriptionally inactive (Cleavinger et al., 1989; Takamiya et al., 1993). Embryonation requires oxygen and after about 48–72 h is accompanied by

CHAPTER 33 – Metabolism and Energetics of Vascular Smooth Muscle

Abstract: This chapter focuses on the metabolism and energetics of vascular smooth muscle (VSM) contractility. In vascular smooth muscle, the metabolic demand of the contractile machinery for ATP is matched by its capacity to resynthesize ATP. The biochemical pathways for ATP synthesis appear similar between mammalian skeletal muscle and VSM, as all the enzymes necessary for glycolysis, the citric acid cycle, and the respiratory transport chain, are present. VSM oxidizes a wide variety of exogenous and endogenous substrates, including glucose, glycogen, fatty acids, and branched chain amino acids. Increases in the rate of aerobic metabolism are correlated very tightly with the levels of maintained isometric tension under a wide variety of conditions. Regulation of oxidative metabolism in VSM may occur through an alternative method, which modulates the activity of mitochondrial dehydrogenase enzymes. Mitochondrial dehydrogenase enzymes include pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and succinate dehydrogenase. These enzymes are responsible for the oxidation of substrates, resulting in the transfer of electrons to either NAD(P) + or FAD + producing NAD(P)H or FADH.