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PubMed1977

Novel enzymic machinery for the metabolism of oxalacetate, phosphoenolpyruvate, and pyruvate in Pseudomonas citronellolis.

The metabolic pathways for the interconversion of oxalacetate, phosphoenolpyruvate, and pyruvate in Pseudomonas citronellolis form an interlocking system (Scheme 1) that would appear to require complex regulatory mechanisms to permit a proper flow of metabolites through the pathways and to prevent futile cycling. Oxalacetate decarboxylase (I in Scheme 1), P-enolpyruvate synthase (II), P-enolpyruvate carboxylase (III), and pyruvate kinase (V) are constitutive enzymes in this organism. Pyruvate carboxylase (VI) is inducible and has its highest activity in cells grown on glucose or lactate, moderate activity in cells grown on acetate, citrate, or glutamate, and virtually no activity in aspartate-grown cells. P-enolpyruvate carboxykinase (IV) was not detected. The presence of these five enzymes in a single cell has not been previously reported. In Scheme 1, three futile cycles are possible: the simultaneous operation of Reactions I and VI; of Reactions II and V; or of I, II, and III. An examination of the regulatory properties of the individual enzymes after partial purification offers support for the hypothesis of an intricate regulatory system. Oxalacetate decarboxylase (I) is inhibited by acetyl-CoA; phosphoenolpyruvate carboxylase (III) is activated by acetyl-CoA and ADP and inhibited by aspartate; phosphoenolpyruvate synthase (II) is inhibited by 5'-AMP and phosphoenolpyruvate; and pyruvate kinase (V) is activated by 5'-AMP and 2 keto, 3-deoxy,6-phosphogluconate and inhibited by ATP. The presence of metabolites with reciprocal but reinforcing functions is noteworthy. As an example, acetyl-CoA both inhibits the breakdown of oxalacetate and stimulates its formation. Only pyruvate carboxylase appears to be regulated by the carbon substrates of the growth medium.

Acetyl Coenzyme A↗
PubMed2004

Autoimmune epitopes: autoepitopes.

The identity of reactants for autoantibodies has been successively refined from whole cellular organelles (immunofluorescence), identified molecules (immunoblot; gene expression libraries), epitope regions (truncated cDNAs; peptide scanning) to contact residues, as described here. Most autoantibodies react with conformational epitopes, in which amino acids distant in the linear sequence come into contiguity by protein folding. Identification of contact sites with the antibody paratope requires particular technologies, crystallography, or antibody screening of phage-displayed random peptide libraries. The latter is illustrated by our studies on the autoepitope for anti-PDC-E2 (AMA) in primary biliary cirrhosis (PBC), anti-GAD65 in type 1 diabetes, and anti-C1 of type II collagen in collagen-induced arthritis. More precise definition of the structure of conformational autoepitopes could (a) clarify controversial aspects of autoimmunity including epitope mimicry, epitope spreading, and molecular spatial relationships between B and T cell autoepitopes, and (b) impact on novel diagnostic and therapeutic (vaccine) molecules.

Animals↗
PubMed1990

Comparison of the Kinetic Behavior toward Pyridine Nucleotides of NAD-Linked Dehydrogenases from Plant Mitochondria.

In this article we compare the kinetic behavior toward pyridine nucleotides (NAD(+), NADH) of NAD(+)-malic enzyme, pyruvate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine decarboxylase extracted from pea (Pisum sativum) leaf and potato (Solanum tuberosum) tuber mitochondria. NADH competitively inhibited all the studied dehydrogenases when NAD(+) was the varied substrate. However, the NAD(+)-linked malic enzyme exhibited the weakest affinity for NAD(+) and the lowest sensitivity for NADH. It is suggested that NAD(+)-linked malic enzyme, when fully activated, is able to raise the matricial NADH level up to the required concentration to fully engage the rotenone-resistant internal NADH-dehydrogenase, whose affinity for NADH is weaker than complex I.

Journal Article↗
PubMed1975

Erythritol catabolism by Brucella abortus.

Cell extracts of Brucella abortus (British 19) catabolized erythritol through a series of phosphorylated intermediates to dihydroxyacetonephosphate and CO-2. Cell extracts required adenosine 5'-triphosphate (ATP), nicotinamide adenine dinucleotide (NAD), Mg2+, inorganic orthophosphate, and reduced glutathione for activity. The first reaction in the pathway was the phosphorylation of mesoerythritol with an ATP-dependent kinase which formed d-erythritol 1-phosphate (d-erythro-tetritol 1-phosphate). d-Erythritol 1-phosphate was oxidized by an NAD-dependent dehydrogenase to d-erythrulose 1-phosphate (d-glycero-2-tetrulose 1-phosphate). B. abortus (US-19) was found to lack the succeeding enzyme in the pathway and was used to prepare substrate amounts of d-erythrulose 1-phosphate. d-Erythritol 1-phosphate dehydrogenase (d-erythro-tetritol 1-phosphage: NAD 2-oxidoreductase) is probably membrane bound. d-Erythrulose 1-phosphate was oxidized by an NAD-dependent dehydrogenase to 3-keto-l-erythrose 4-phosphate (l-glycero-3-tetrosulose 4-phosphate) which was further oxidized at C-1 by a membrane-bound dehydrogenase coupled to the electron transport system. Either oxygen or nitrate had to be present as a terminal electron acceptor for the oxidation of 3-keto-l-erythrose 4-phosphate to 3-keto-l-erythronate 4-phosphate (l-glycero-3-tetrulosonic acid 4-phosphate). The beta-keto acid was decarboxylated by a soluble decarboxylase to dihydroxyacetonephosphate and CO-2. Dihydroxyacetonephosphate was converted to pyruvic acid by the final enzymes of glycolysis. The apparent dependence on the electron transport system of erythritol catabolism appears to be unique in Brucella and may play an important role in coupling metabolism to active transport and generation of ATP.

Adenosine Triphosphate↗
PubMed1989

Physically associated enzymes produce and metabolize 2-hydroxy-2,4-dienoate, a chemically unstable intermediate formed in catechol metabolism via meta cleavage in Pseudomonas putida.

The meta-cleavage pathway of catechol is a major mechanism for degradation of aromatic compounds. In this pathway, the aromatic ring of catechol is cleaved by catechol 2,3-dioxygenase and its product, 2-hydroxymuconic semialdehyde, is further metabolized by either a hydrolytic or dehydrogenative route. In the dehydrogenative route, 2-hydroxymuconic semialdehyde is oxidized to the enol form of 4-oxalocrotonate by a dehydrogenase and then further metabolized to acetaldehyde and pyruvate by the actions of 4-oxalocrotonate isomerase, 4-oxalocrotonate decarboxylase, 2-oxopent-4-enoate hydratase, and 4-hydroxy-2-oxovalerate aldolase. In this study, the isomerase, decarboxylase, and hydratase encoded in the TOL plasmid pWW0 of Pseudomonas putida mt-2 were purified and characterized. The 28-kilodalton isomerase was formed by association of extremely small identical protein subunits with an apparent molecular weight of 3,500. The decarboxylase and the hydratase were 27- and 28-kilodalton polypeptides, respectively, and were copurified by high-performance-liquid chromatography with anion-exchange, hydrophobic interaction, and gel filtration columns. The structural genes for the decarboxylase (xylI) and the hydratase (xylJ) were cloned into Escherichia coli. The elution profile in anion-exchange chromatography of the decarboxylase and the hydratase isolated from E. coli XylI+XylJ- and XylI-XylJ+ clones, respectively, were different from those isolated from XylI+ XylJ+ bacteria. This suggests that the carboxylase and the hydratase form a complex in vivo. The keto but not the enol form of 4-oxalocrotonate was a substrate for the decarboxylase. The product of decarboxylation was 2-hydroxypent-2,4-dienoate rather than its keto form, 2-oxopent-4-enoate. The hydratase acts on the former but not the latter isomer. Because 2-hydroxypent-2,4-dienoate is chemically unstable, formation of a complex between the decarboxylase and the hydratase may assure efficient transformation of this unstable intermediate in vivo.

Carboxy-Lyases↗
PubMed1985

Pyruvoyl-dependent histidine decarboxylases. Mechanism of cleavage of the proenzyme from Lactobacillus buchneri.

When Lactobacillus buchneri was grown in the presence of [hydroxyl-18O]serine and pyridoxamine, no 18O was found in its histidine decarboxylase (HisDCase). However, when pyridoxamine was omitted from the growth medium, the labeled serine was incorporated into the HisDCase without dilution. Internal serine residues of the enzyme contained 18O only in their hydroxyl group, while the COOH-terminal serine of the beta chain of HisDCase contained equal amounts of 18O in both its hydroxyl and carboxyl group. This enzyme, like the HisDCase from Lactobacillus 30a (Recsei, P. A., Huynh, Q. K., and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 973-977), therefore, arises by nonhydrolytic serinolysis of its proenzyme. This result, together with comparative sequence data (Huynh, Q. K., and Snell, E. E. (1985) J. Biol. Chem. 260, 2798-2803), makes it highly probable that all of the pyruvoyl-dependent HisDCases arise by a similar mechanism from inactive proenzymes.

Amino Acid Sequence↗
PubMed2001

Lactobacillus casei and Lactobacillus plantarum initiate catabolism of methionine by transamination.

AIMS: To study the ability of Lactobacillus casei and Lact. plantarum strains to convert methonine to cheese flavour compounds. METHODS AND RESULTS: Strains were assayed for methionine aminotransferase and lyase activities, and amino acid decarboxylase activity. About 25% of the strains assayed showed methionine aminotransferase activity. The presence of glucose in the reaction mixture increased conversion of methionine to 4-methylthio-2-ketobutanoate (KMBA) and 4-methylthio-2-hydroxybutanoate (HMBA) in all strains. The methionine aminotransferase activity in Lact. plantarum and Lact. casei showed variable specificity for the amino group acceptors glyoxylate, ketoglutarate, oxaloacetate and pyruvate. None of the strains showed methionine lyase or glutamate and methionine decarboxylase activities. CONCLUSION: The presence of amino acid converting enzymes in lactobacilli is strain specific. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work suggest that lactobacilli can be used as adjuncts for flavour formation in cheese manufacture.

Carbon-Sulfur Lyases↗
PubMed1981

Bacterial degradation of 3,4,5-trimethoxyphenylacetic and 3-ketoglutaric acids.

When grown at the expense of 3,4,5-trimethoxyphenylacetic acid, a species of Arthrobacter readily oxidized 3,4-dihydroxy-5-methoxyphenylacetic acid, but other structurally related aromatic acids were oxidized only slowly. Cell extracts contained a dioxygenase for 3,4-dihydroxy-5-methoxyphenylacetate, and the corresponding trihydroxy acid, which was not attacked by the enzyme, inhibited oxidation of this ring-fission substrate. Cell suspensions did not release carbon dioxide from 3,4-[methoxyl-14C]dihydroxy-5-methoxyphenylacetate but accumulated 1 mol of methanol per mol of 3,4,5-trimethoxyphenylacetate oxidized. A cell extract converted the ring-fission substrate into stoichiometric amounts of pyruvate and acetoacetate, formed from 3-ketoglutarate by the action of an induced decarboxylase. 3-Ketoglutaric acid served as sole source of carbon for many soil isolates.

Acetoacetates↗
PubMed2001

Hypoxia in vivo decreases peroxisome proliferator-activated receptor alpha-regulated gene expression in rat heart.

We tested the hypothesis that hypoxia decreases PPARalpha-regulated gene expression in heart muscle in vivo. In two rat models of systemic hypoxia (cobalt chloride treatment and iso-volemic hemodilution), transcript levels of PPARalpha and PPARalpha-regulated genes (pyruvate dehydrogenase kinase 4 (PDK4), muscle carnitine palmitoyltransferase-I (mCPT-I), and malonyl-CoA decarboxylase (MCD)) were measured using real-time quantitative RT-PCR. Data were normalized to the housekeeping gene beta-actin. Atrial natriuretic factor (ANF) and pyruvate dehydrogenase kinase 2 (PDK2), which are not regulated by PPARalpha, served as controls. CoCl(2) treatment decreased PPARalpha, PDK4, mCPT-I, and MCD mRNA levels. Iso-volemic anemia also caused a significant decrease in PPARalpha, PDK4, and MCD mRNA levels. Transcript levels of mCPT-I showed a slight, but not significant decrease (P = 0.08). Gene expression of beta-actin, ANF, and PDK2 did not change with either CoCl(2) treatment nor with anemia. Myocardial PPARalpha-regulated gene expression is decreased in two models of hypoxia in vivo. These results suggest a transcriptional mechanism for decreased fatty oxidation and increased reliance of the heart for glucose during hypoxia.

Animals↗
PubMed1980

The relationship of 4-aminobutyric acid metabolism to ammoniagenesis in renal cortex.

Mitochondrial 4-aminobutyrate aminotransferase in rat kidney can utilize pyruvate as the acceptor for the amino group of 4-aminobutyrate. Renal 4-aminobutyrate aminotransferase activity at saturating equimolar concentration of 4-aminobutyrate and 5 mM pyruvate is 42.8 +/- 2.5 mumol/g protein per h (mean +/- S.E.M.) or 70% of 4-aminobutyrate aminotransferase activity with equimolar alpha-ketoglutarate. 4-Aminobutyrate aminotransferase in brain does not transaminate with pyruvate. Since pyruvate is an important mitochondrial metabolite in kidney, net disposal of glutamate via the 4-aminobutyrate pathway is possible. The renal 4-aminobutyrate pathway in the rat has other distinctive features when compared with the pathway in rat brain. Most inhibitors of rat neuronal glutamate decarboxylase were ineffective against the renal form of the enzyme, but 20 mM semicarbazide inhibited the latter form by 80% (P < 0.001) in vitro and reduced renal 4-aminobutyrate content by 75% (P < 0.001) in vivo. In the presence of 20 mM semicarbazide, ammoniagenesis by rat renal cortex slices incubated in 1 mM glutamine was inhibited 26% (P < 0.01). Semicarbazide was proportionately less effective (15% inhibition) when ammonia-genesis was stimulated (+243%) in slices prepared from chronically acidotic animals, and was no deterrant to ammoniagenesis when non-acidotic slices were incubated in supraphysiologic concentrations of 10 mM glutamine. We conclude that whereas integrity of the renal 4-aminobutyrate pathway may contribute to glutamate disposal and thus ammoniagenesis under physiologic conditions, the pathway is a passive participant in the overall process of ammoniagenesis.

4-Aminobutyrate Transaminase↗
PubMed1999

Oxaloacetate decarboxylase from Pseudomonas stutzeri: purification and characterization.

Oxaloacetate decarboxylase (OXAD), the enzyme that catalyzes the decarboxylation of oxaloacetate to pyruvic acid and carbon dioxide, was purified 245-fold to homogeneity from Pseudomonas stutzeri. The three-step purification procedure comprised anion-exchange chromatography, metal-chelate affinity chromatography, and biomimetic-dye affinity chromatography. Estimates of molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native high-performance gel-filtration liquid chromatography were, respectively, 63 and 64 kDa, suggesting a monomeric protein. OXAD required for maximum activity divalent metal cations such as Mn2+ and Mg2+ but not monovalent cations. The enzyme is not inhibited by avidin, but is competitively inhibited by adenosine 5'-diphosphate, acetic acid, phosphoenolpyruvate, malic acid, and oxalic acid. Initial velocity, product inhibition, and dead-end inhibition studies suggested a rapid-equilibrium ordered kinetic mechanism with Mn2+ being added to the enzyme first followed by oxaloacetate, and carbon dioxide is released first followed by pyruvate. Inhibition data as well as pH-dependence profiles and kinetic parameters are reported and discussed in terms of the mechanism operating for oxaloacetate decarboxylation.

Bicarbonates↗
PubMed1997

Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z.

Actinobacillus sp. 130Z fermented glucose to the major products succinate, acetate, and formate. Ethanol was formed as a minor fermentation product. Under CO2-limiting conditions, less succinate and more ethanol were formed. The fermentation product ratio remained constant at pH values from 6.0 to 7.4. More succinate was produced when hydrogen was present in the gas phase. Actinobacillus sp. 130Z grew at the expense of fumarate and l-malate reduction, with hydrogen as an electron donor. Other substrates such as more-reduced carbohydrates (e.g., d-sorbitol) resulted in higher succinate and/or ethanol production. Actinobacillus sp. 130Z contained the key enzymes involved in the Embden-Meyerhof-Parnas and the pentose-phosphate pathways and contained high levels of phosphoenolpyruvate (PEP) carboxykinase, malate dehydrogenase, fumarase, fumarate reductase, pyruvate kinase, pyruvate formate-lyase, phosphotransacetylase, acetate kinase, malic enzyme, and oxaloacetate decarboxylase. The levels of PEP carboxykinase, malate dehydrogenase, and fumarase were significantly higher in Actinobacillus sp. 130Z than in Escherichia coli K-12 and accounted for the differences in succinate production. Key enzymes in end product formation in Actinobacillus sp. 130Z were regulated by the energy substrates.

Actinobacillus↗
PubMed1999

pH studies on the mechanism of the pyridoxal phosphate-dependent dialkylglycine decarboxylase.

The pH dependence of the steady-state kinetic parameters for the dialkylglycine decarboxylase-catalyzed decarboxylation-dependent transamination between 2-aminoisobutyrate (AIB) and pyruvate is presented. The pH dependence of methylation and DTNB modification reactions, and spectroscopic properties, is used to augment the assignment of the kinetic pKa's to specific ionizations. The coincidence of pKa values (approximately 7.4) observed in kcat/KAIB, 1/KAIB, Kis for pyruvate, KPLP, and in absorbance and fluorescence titrations demonstrates that AIB is not a sticky substrate. It furthermore suggests that the decarboxylation step, or a conformational isomerization preceding it, limits the rate of the overall catalytic cycle. Coexisting, kinetically distinguishable conformers of DGD-PLP, originating from an alkali metal ion binding site, were previously demonstrated at pH 8.2 for DGD-PLP (Zhou, X., Toney, M. D. Biochemistry 37, 5761-5769). The pKa value of approximately 8.8 observed in kcat, kcat/KAIB, Kd for K+, spectrometric titrations, and the reaction of DGD-PLP with DTNB is tentatively assigned to the conformational change interconverting the two enzyme forms previously characterized. Three pKa's are observed in pH titrations of the DGD-PLP coenzyme absorbance. Individual spectra for the four ionization states are deconvoluted by fitting log-normal curves. All four ionization states have both ketoenamine and enolimine tautomers present. This and a review of spectral data in the literature lead to the conclusion that the pKa of approximately 7.4, which gives the largest spectral changes and controls kcat/KAIB, is not deprotonation of the aldimine nitrogen. Rather, it must be an active site residue whose ionization alters the ratio between ketoenamine and enolimine tautomers.

Binding Sites↗
PubMed1999

Transcription factor GCN4 for control of amino acid biosynthesis also regulates the expression of the gene for lipoamide dehydrogenase.

The yeast LPD1 gene encoding lipoamide dehydrogenase is subject to the general control of amino acid biosynthesis mediated by the GCN4 transcription factor. This is striking in that it demonstrates that GCN4-mediated regulation extends much farther upstream than simply to the direct pathways for amino acid and purine biosynthesis. In yeast, lipoamide dehydrogenase functions in at least three multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase (which function in the entry of pyruvate into, and metabolism via, the citric acid cycle) and glycine decarboxylase. When wild-type cells were shifted from growth on amino acid-rich to amino acid-deficient medium, the expression of lipoamide dehydrogenase was induced approx. 2-fold. In a similar experiment no such induction was observed in isogenic gcn4 mutant cells. Northern analysis indicated that amino acid starvation affected levels of the LPD1 transcript. In the upstream region of LPD1 are three matches to the consensus for control mediated by GCN4. Directed mutagenesis of each site, and of all combinations of sites, suggests that only one site might be important for the general control response under the conditions tested. Gel-retardation analysis with GCN4 protein synthesized in vitro has indicated that GCN4 can bind in vitro to at least two of the consensus motifs.

Amino Acid Sequence↗
PubMedDate not supplied

[Decarboxylase activity and biosynthetic processes in Saccharomyces carlsbergensis exposed to light].

Light at a wavelength from 410 to 520 nm was found to stimulate the activity of decarboxylase, protein biosynthesis and the yield of biomass in Saccharomyces carlsbergensis. Pyruvate decarboxylation yielding a substrate for the di- and tricarboxylic acid cycle is a limiting step in the metabolism of the yeast. Light can activate the process, thus eliminating the substrate deficiency of the cycle and increasing the rate of anabolic processes.

Carboxy-Lyases↗
PubMedDate not supplied

Isolation, properties and role in progesterone biosynthesis of cytosolic malic enzyme from human term placenta.

Cytosolic malic enzyme (L-malate: NADP oxidoreductase decarboxylating, EC I. I. I.40) has been isolated and purified from postmitochondrial supernatant of human term placenta by ammonium sulphate fractionation, chromatography on diethylaminoethyl- (DEAE-)cellulose, Sepharose 6B, ADP-Sepharose 4B and Ultrogel AcA-34 to apparent homogeneity as judged from polyacrylamide gel electrophoresis (PAGE). The specific activity of the purified enzyme was 24.0 mumol X min-1 X mg-1 protein, which corresponds to about 7500-fold purification. The molecular weight of the native enzyme was determined by gel filtration to be about 250,000. Sodium dodecyl sulphate- (SDS-)PAGE showed one polypeptide band of molecular weight 63,000. It appears that the native protein is a tetramer composed of subunits of identical molecular weight. The isoelectric point of the purified malic enzyme was pH 5.55. The enzyme was shown to carboxylate pyruvate in the presence of high concentrations of pyruvate and bicarbonate at about 80 per cent of the rate of the forward reaction. The optimum pH for the carboxylation reaction was pH 7.3, and that for the decarboxylation reaction varied with malate concentration. The Km values, determined at pH 7.2, for malate, NADP+, Mn2+, and Mg2+ were 81 microM, 10 microM, 2.5 microM and 0.6 mM, respectively. The Km values for pyruvate, NADPH and bicarbonate were 4 mM, 25 microM and 20 mM, respectively. The enzyme converted malate to pyruvate (at pH 6.3) in the presence of 5 mM NAD+ at approximately 80 per cent of the maximum rate with NADP+. It exhibited oxaloacetate decarboxylase activity at about 10 per cent of the rate of oxidative decarboxylation of malate (with NADP+ as coenzyme) and pyruvate reductase activity at about 4 per cent of the rate of oxidative decarboxylation of malate with NADP+. The oxidative decarboxylation of malate was inhibited by malate at lower values of pH of incubation medium. This inhibition gradually decreased as the pH of the incubation medium increased. No inhibition was observed at pH 8.2. The addition of purified cytoplasmic malic enzyme, pyruvate, bicarbonate and NADPH generating system (consisting of NADP+, glucose 6-phosphate, glucose 6-phosphate dehydrogenase) stimulated about twofold progesterone biosynthesis by the isolated human placental mitochondria. This stimulation was abolished by arsenite and fluorocitrate. A possible role for the cytosolic malic enzyme in the regulation of progesterone biosynthesis in human placenta is discussed.

Electrophoresis, Polyacrylamide Gel↗