[Mitochondrial myopathy with increased blood lactate and pyruvate levels among siblings in familial thiamine deficiency].
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Adolescent↗
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Results for “Pyruvate Decarboxylase”
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The stimulation of hepatic gluconeogenesis by acetoacetate precursors. A role for the monocarboxylate translocator.
The regulation of the gluconeogenic pathway from the 3-carbon precursors pyruvate, lactate, and alanine was investigated in the isolated perfused rat liver. Using pyruvate (less than 1 mM), lactate, or alanine as the gluconeogenic precursor, infusion of the acetoacetate precursors oleate, acetate, or beta-hydroxybutyrate stimulated the rate of glucose production and, in the case of pyruvate (less than 1 mM), the rate of pyruvate decarboxylation. alpha-Cyanocinnamate, an inhibitor of the monocarboxylate transporter, prevented the stimulation of pyruvate decarboxylation and glucose production due to acetate infusion. With lactate as the gluconeogenic precursor, acetate infusion in the presence of L-carnitine stimulated the rate of gluconeogenesis (100%) and ketogenesis (60%) without altering the tissue acetyl-CoA level usually considered a requisite for the stimulation of gluconeogenesis by fatty acids. Hence, our studies suggest that gluconeogenesis from pyruvate or other substrates which are converted to pyruvate prior to glucose synthesis may be limited or controlled by the rate of entry of pyruvate into the mitochondrial compartment on the monocarboxylate translocator.
3-Hydroxybutyric Acid↗
Evidence for separate enzymes of pyruvate decarboxylation and pyruvate synthesis in soluble extracts of Clostridium pasteurianum.
Additional evidence to that already presented (Sauer, F. D., Bush, R. S., and Stevenson, I. L. (1976) Biochim. Biophys. Acta 445, 518-520) suggests that pyruvate-ferredoxin oxidoreductase isolated from Clostridium pasteurianum consists of two separate enzymes: (a) pyruvate lyase, which catalyzes the CoA and electron acceptor-dependent decarboxylation of pyruvate, and (b) pyruvate synthase, which catalyzes the reduced ferredoxin-dependent carboxylation of acetyl-CoA to pyruvate. The enzymes separated on Sephadex G-200 and with acrylamide gel electrophoresis but complete separation of one enzyme free of the other was not achieved. Extensive purification procedures were not used because both enzymes are unstable. The results confirm published reports that pyruvate lyase contains thiamin and a chromophore which participates in electron transfer. Pyruvate synthase, however, did not appear to be a thiamin enzyme and there was no evidence to indicate participation of an enzyme chromophore in the pyruvate synthase reaction.
Carboxy-Lyases↗
Purification and properties of Escherichia coli 4'-phosphopantothenoylcysteine decarboxylase: presence of covalently bound pyruvate.
4'-Phosphopantothenoylcysteine decarboxylase was purified 900-fold from Escherichia coli B with an overall yield of 6%. The enzyme migrates as a single band with a molecular weight of 35,000 +/- 3000 in 10% polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has an apparent molecular weight of 146,000 +/- 9000 as determined by a gel exclusion column. At pH 7.6 and 25 degrees C, Km = 0.9 mM and Vmax = 600 nmol/(min X mg of protein). The pH optimum for Vmax is between 7.5 and 7.7. Hydroxylamine, phenylhydrazine, potassium cyanide, and sodium borohydride as well as pyridoxal phosphate and pyridoxal inactivated the enzyme. The enzyme contains covalently bound pyruvate as suggested by the isolation of [3H]lactate and pyruvate from [3H]NaBH4-reduced enzyme and native enzyme, respectively. One mole of [3H]lactate was isolated per 39,000 g of [3H]NaBH4-reduced and completely inactivated enzyme, and 1 mol of pyruvate was isolated per 31,000 +/- 4000 g of native enzyme. Mild base treatment released lactate and pyruvate from the reduced and the native enzymes, respectively, suggesting the pyruvate is attached to the enzyme by an ester bond. These findings are in accord with similar results obtained with the horse liver enzyme (R. Scandurra, personal communication). The presence of covalently bound pyruvate in the bacterial and mammalian enzymes suggests that pyruvate plays a major role in the mechanism of action.
Binding Sites↗
Phosphonate analogues of pyruvate. Probes of substrate binding to pyruvate oxidase and other thiamin pyrophosphate-dependent decarboxylases.
A number of enzymes catalyze the removal of carbon dioxide from pyruvate through covalent participation of the coenzyme thiamin pyrophosphate. The conversions of the decarboxylated adduct, hydroxyethyl thiamin pyrophosphate, to subsequent products distinguishes the function of these enzymes. Acetaldehyde is produced by pyruvate decarboxylase, acetic acid by pyruvate oxidase and acetyl coenzyme A by pyruvate dehydrogenase. Differences and details of steps prior to decomposition of hydroxyethyl thiamin pyrophosphate can be evaluated through the use of two substrate analogues, methyl acetylphosphonate and acetylphosphonate. Methyl acetylphosphonate and acetylphosphonate are competitive inhibitors toward pyruvate with Escherichia coli pyruvate oxidase and E. coli pyruvate dehydrogenase but the value of the Ki for the oxidase is more than three orders of magnitude higher than for the dehydrogenase. Yeast pyruvate decarboxylase is not inhibited at all under the same conditions. The binding of methyl acetylphosphonate results in ligand-induced changes in the near ultraviolet circular dichorism spectrum of the oxidase. This spectral perturbation is only seen in the presence of the cofactor, thiamin pyrophosphate, strongly suggesting that the inhibitor is binding at the same site as the substrate, pyruvate, on the enzyme. Kinetic data suggest that lipid activators of pyruvate oxidase increase the affinity of the enzyme for pyruvate and its analogues.
Acetaldehyde↗
Covalently bound pyruvate in phosphopantothenoylcysteine decarboxylase from horse liver.
Horse liver phosphopantothenoylcysteine decarboxylase (EC 4.1.1.36) incorporates nonexchangeable tritium from borotritide with a decrease of the activity. Substrate prevents both tritium incorporation and the decrease in activity. Acid and base hydrolysis of the tritiated protein releases labeled lactate identified by high-voltage paper electrophoresis, paper chromatography and silicic acid chromatography. These results indicate the presence of pyruvate covalently bound through an ester linkage to phosphopantothenoylcysteine decarboxylase which is then another example of a mammalian enzyme in which pyruvate is involved in a catalytic activity.
Amino Acid Transport Systems, Neutral↗
Stereochemistry and kinetic isotope effects in the decarboxylation of S-adenosylmethionine catalyzed by the pyruvyl enzyme, S-adenosylmethionine decarboxylase.
S-Adenosyl-5'-3-methylthio[1-3H]propylamine was prepared by decarboxylation of S-adenosylmethionine in tritiated water, catalyzed by S-adenosylmethionine decarboxylase from Escherichia coli. Degradation of this product to 3-methylthio[1-3H]propylamine followed by oxidation in a coupled assay system involving pea seedling amine oxidase, catalase, and aldehyde dehydrogenase indicated less than 4% of total counts in water and greater than 75% in NADH plus NAD+. These results allow the assignment of tritium in 3-methylthiopropylamine to the 1-R configuration, establishing that S-adenosylmethionine decarboxylase, a pyruvate-containing decarboxylase, operates via a retentive mode. Tabulation of the available stereochemical results for two pyruvate-containing and three pyridoxal phosphate-dependent amino acid decarboxylases indicates that stereochemistry has been conserved both within each class of amino acid decarboxylases and between structurally distinct classes of decarboxylases. Comparison of the specific activity of 3-methylthio[1-3H]propylamine to the tritiated water employed in the decarboxylation reaction leads to a primary tritium isotope effect of 4.5 under conditions of substrate concentration far in excess of Km. Although a portion of the tritium in 3-methylthiopropylamine could have arisen through an enzyme-catalyzed exchange into decarboxylated adenosylmethionine (leading to an underestimate of the kinetic tritium isotope effect), we are unable to detect enzymatic loss of tritium from decarboxylated S-adenosylmethionine. The magnitude of the observed isotope effect is discussed in the context of a kinetically significant exchange of an active site residue with solvent in the present of enzyme-bound S-adenosylmethionine.
Adenosylmethionine Decarboxylase↗
Pyruvate cycling involving possible oxaloacetate decarboxylase activity.
With high concentrations of pyruvate as substrate for hepatocytes from fasted rats, high rates of cycling between pyruvate and the dicarboxylic acids occur, as shown isotopically. This rate of cycling is adequate to account for the hydrogen translocation from the mitochondria to the cytosol to furnish NADH for lactate formation. Addition of sufficiently high concentrations of mercaptopicolinate to block almost completely glucose formation from pyruvate, depresses isotopic cycling and lactate formation by only about 50-75%. Under some conditions, when the normal phosphoenolpyruvate carboxykinase activity is inhibited, cytosolic oxaloacetate may be decarboxylated directly to pyruvate, possibly via the decarboxylase activity of phosphoenolpyruvate carboxykinase.
Animals↗
Glucose/citrate cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with impaired alpha-acetolactate decarboxylase.
The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in alpha-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The alpha-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, alpha-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on alpha-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired alpha-acetolactate decarboxylase activity accumulated alpha-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.
Biotechnology↗
Bioelectrochemical determination of citric acid in real samples using a fully automated flow injection manifold.
An enzymic method for the determination of citric acid in fruits, juices and sport drinks is proposed. The method is based on the action of the enzymes citrate lyase, oxaloacetate decarboxylase and pyruvate oxidase, which convert citric acid into H2O2 with the latter being monitored amperometrically with a H2O2 probe. The enzymes pyruvate oxidase and oxaloacetate decarboxylase were immobilized. A multi-membrane system, consisting of a cellulose acetate membrane for the elimination of interferants, an enzymic membrane and a protective polycarbonate membrane were placed on a Pt electrode and used with a fully automated flow injection manifold. Several parameters were optimized, resulting in a readily constructed and reproducible biosensor. Interference from various compounds present in real samples was minimized. Calibration graphs were linear over the range 0.01-0.9 mM pyruvate, 0.015-0.6 mM oxaloacetate and 0.015-0.5 mM citrate. The throughput was 30 samples h-1 with an RSD of 1.0% (n = 8); the mean relative error was 2.4% compared with a standard method. The recovery was 96-104%. A 8-10% loss of the initial activity of the sensor was observed after 100-120 injections.
Beverages↗
Mechanism of pigeon liver malic enzyme modification of histidyl residues by ethoxyformic anhydride.
Incubation of malic enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) with ethoxyformic anhydride caused the time-dependent loss of its ability to catalyze reactions requiring the nucleotide cofactor NADP+ or NADPH, such as the oxidative decarboxylase, the NADP+ - stimualted oxalacetate decarboxylase, the pyruvate reductase, and the pyruvate-medium proton exchange activities. Similar loss of oxidative decarboxylase and pyruvate reductase activities was affected by photo-oxidation in the presence of rose bengal. The inactivation of oxidative decarboxylase activity by ethoxyformic anhydride was accompanied by the reaction of greater than or equal to 2.3 histidyl residues per enzyme site and was strongly inhibited by NADP+. Ethoxyformylation also impaired the ability of malic enzyme to bind NADP+ or NADPH. These results support the involvement of histidyl residue(s) at the nucleotide binding site of malic enzyme.
Animals↗
Studies on the mechanism of formation of the pyruvate prosthetic group of phosphatidylserine decarboxylase from Escherichia coli.
Phosphatidylserine decarboxylase from Escherichia coli uses a pyruvate group as the enzyme cofactor (Satre, M., and Kennedy, E. P. (1978) J. Biol. Chem. 253, 479-483). Comparison of the DNA sequence of the psd gene with the partial amino acid sequence of the mature gene product suggests that the two nonidentical subunits of the mature enzyme are formed by cleavage of a proenzyme resulting in the conversion of Ser-254 to an amino-terminal pyruvate residue (Li, Q.-X., and Dowhan, W. (1988) J. Biol. Chem. 263, 11516-11522). The cleavage of the wild-type proenzyme occurs rapidly with a half-time on the order of 2 min. When Ser-254 is changed to cysteine (S254C), threonine (S254T), or alanine (S254A) by site-directed mutagenesis, the rate of processing of the proenzyme and the production of the functional enzyme are drastically affected. Proenzymes with S254C or S254T are cleaved with a half-time of around 2-4 h while the S254A proenzyme does not undergo processing. The reduced processing rate for the mutant proenzymes is consistent with less of the functional enzyme being made. Mutants encoding the S254C and S254T protein produce 16 and 2%, respectively, of the activity of the wild-type allele but can still complement a temperature-sensitive mutant in the psd locus. There is no detectable activity or complementation observed with the S254A protein. These results are consistent with the hydroxyl group of Ser-254 playing a critical role in the cleavage of the peptide bond between Gly-253 and Ser-254 of the prophosphatidylserine decarboxylase and support the mechanism proposed by Snell and coworkers (Recsei and Snell (1984) Annul Rev. Biochem. 53, 357-387) for the formation of the prosthetic group of pyruvate-dependent decarboxylases.
Carboxy-Lyases↗
[Localization of pyruvic acid in the histidine decarboxylase of Micrococcus sp. n].
A fragment containing the pyruvic acid residue is isolated from hymotryptic hydrolyzate of alpha-polypeptide chain of carboxymethylated histidine decarboxylase (HD) 14C-phenylhydrasone. The pyruvate residue is estimated to be bind with alpha-amino group of N-terminal phenylalanine in alpha-polypeptide chain. N-terminal alanine is found in HD alpha-polypeptide chain after its reductive amination. It makes possible to determine the estimation of N-terminal amino acid sequence in HD alpha-chain.
Carboxy-Lyases↗
Inhibition of enzymes of polyamine biosynthesis by substrate-like O-substituted hydroxylamines.
The biogenic amines spermine, spermidine, and putrescine are essential factors of cell growth and differentiation. To inhibit pyridoxal-5'-phosphate dependent ornithine decarboxylase and pyruvate dependent S-adenosylmethionine decarboxylase, key enzymes of polyamine biosynthesis, a system of substrate-like O-substituted hydroxylamines is suggested. The best of these compounds were active at nanomolar concentrations. High potency and specificity of this type of inhibitors are discussed in terms of structural similarity of E-I and E-S complexes.
Adenosylmethionine Decarboxylase↗
Identification of the gene encoding sulfopyruvate decarboxylase, an enzyme involved in biosynthesis of coenzyme M.
The products of two adjacent genes in the chromosome of Methanococcus jannaschii are similar to the amino and carboxyl halves of phosphonopyruvate decarboxylase, the enzyme that catalyzes the second step of fosfomycin biosynthesis in Streptomyces wedmorensis. These two M. jannaschii genes were recombinantly expressed in Escherichia coli, and their gene products were tested for the ability to catalyze the decarboxylation of a series of alpha-ketoacids. Both subunits are required to form an alpha(6)beta(6) dodecamer that specifically catalyzes the decarboxylation of sulfopyruvic acid to sulfoacetaldehyde. This transformation is the fourth step in the biosynthesis of coenzyme M, a crucial cofactor in methanogenesis and aliphatic alkene metabolism. The M. jannaschii sulfopyruvate decarboxylase was found to be inactivated by oxygen and reactivated by reduction with dithionite. The two subunits, designated ComD and ComE, comprise the first enzyme for the biosynthesis of coenzyme M to be described.
Amino Acid Sequence↗
Lysine 213 and histidine 233 participate in Mn(II) binding and catalysis in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.
Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO2 and plays a key role in gluconeogenesis. This enzyme also has oxaloacetate decarboxylase and pyruvate kinase-like activities. Mutations of PEP carboxykinase have been constructed where the residues Lys213 and His233, two residues of the putative Mn2+ binding site of the enzyme, were altered. Replacement of these residues by Arg and by Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn2+ binding affinity. Lower PEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against urea denaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in V(max) for the primary reaction relative to that for the wild-type enzyme. V(max) values for the oxaloacetate decarboxylase and pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in the mutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn2+ and PEP. The results are consistent with Lys213 and His233 being at the Mn2+ binding site of S. cerevisiae PEP carboxykinase and the Mn2+ affecting the PEP interaction. The different effects of mutations in V(max) for the main reaction and the secondary activities suggest different rate-limiting steps for these reactions.
Amino Acid Substitution↗
Properties of the branched-chain 2-hydroxy acid/2-oxo acid shuttle in mouse spermatozoa.
Operation of the branched-chain 2-hydroxy acid/2-oxo acid shuttle for the transfer of reducing equivalents in mitochondria of mouse spermatozoa was studied in vitro in reconstituted systems. Results show that the branched-chain 2-oxo acids within the mitochondria are offered several metabolic pathways. (a) Decarboxylation: mouse sperm mitochondria possess high branched-chain 2-oxo acid decarboxylase activity. (b) Recycling to the cytosol by using a transport system which can be inhibited by alpha-cyano-3-hydroxycinnamate and pH 6.8. (c) Transamination to the corresponding amino acids: experiments presented indicate that leucine formed from 4-methyl-2-oxopentanoate may pass to the external phase, re-initiating the cycle. These two last possibilities would allow autocatalytic operation of the shuttle. The branched-chain 2-hydroxy acids apparently do not utilize the monocarboxylate carrier to penetrate the mitochondria.
Animals↗
[Effect of C-4'-modification of thiamine pyrophosphate on its coenzyme activity in the oxidative decarboxylation of pyruvic acid reaction].
Interaction was studied between pyruvate dehydrogenase (EC 1.2.4.1) and C-4'-substituted analogs of thiaminpyrophosphate, 4'-N (CH3)-TPP, 4'-N(CH3)2-TPP and OH-TPP. None of these analogs was found to replace TPP during the reduction of NAD and 2.6-dichlorophenol-indophenol as well as pyruvate decarboxylation. The decarboxylase activity of the pyruvate dehydrogenase component isolated from the pyruvate dehydrogenase complex was determined according to the 14CO2 yield and production of 2-C-oxoethyl-TPP using 1-14C-pyruvate and 2-14C-pyruvate, as substrates, respectively. All the analogs were found to competitively inhibit pyruvate dehydrogenase, Ki values for 4'-N(CH3)-TPP, 4'-N(CH3)2-TPP and 4'-OH-TPP being 4.1 X 10(-5) M, 8.5 X 10(-5) M and 2.9 X 10(-6) M, respectively; Km values for TPP was equal to 1-2 X 10(-7) M. It is assumed that the analogs of the holoenzymic complex formed by the pyruvate dehydrogenase component of the pyruvate dehydrogenase complex with mono-, dimethyl-TPP and oxo-TPP do not bind the substrate.
Animals↗