Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role.

The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.     

Distribution of metabolites between the cytosolic and mitochondrial compartments of hepatocytes isolated from fed rats

In isolated hepatocytes from normal fed rats, the subcellular distribution of malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH has been determined by a modified digitonin method. Incubation with various substrates (lactate, pyruvate, alanine, oleate, oleate plus lactate, ethanol and aspartate) markedly changed the total cellular amounts of metabolites, but their distribution between the cytosolic and mitochondrial compartments was kept fairly constant. In the presence of lactate, pyruvate or alanine, about 90% of cellular aspartate, malate and oxaloacetate, and 50% of citrate was located in the cytosol. The changes in acetyl-CoA in the cytosol were opposite to those in the mitochondrial space, the sum of both remaining nearly constant. The mitochondrial acetyl-CoA/CoASH ratio ranged from 0.3-0.9 and was positively correlated with the rate of ketone body formation. The mitochondrial/cytosolic (m/c) concentration gradients for malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH averaged from hepatocytes under different substrate conditions were determined to be 1.0, 8.8, 1.6, 2.2, 0.5, 0.7, 13 and 40, respectively. From the distribution of citrate, a pH difference of 0.3 across the inner mitochondrial membrane was calculated, yet lower values resulted from the m/c gradients of 2-oxoglutarate, glutamate and malate. The mass action ratios for citrate synthase and mitochondrial aspartate aminotransferase have been calculated from the metabolite concentrations measured in the mitochondrial pellet fraction. A comparison with the respective equilibrium constants indicates that in intact hepatocytes, neither enzyme maintains its reactants at equilibrium. On the assumption that mitochondrial malate dehydrogenase and 3-hydroxybutyrate dehydrogenase operate near equilibrium, the concentration of free oxaloacetate appears to be 0.3-2 micron, depending on the substrate used. Plotting the calculated free mitochondrial oxaloacetate concentration against the citrate concentration measured in the mitochondrial pellet yielded a hyperbolic saturation curve, from which an apparent Km of citrate synthase for oxaloacetate in the intact cells of 2 micron can be derived, which is comparable to the value determined with purified rat liver citrate synthase. The results are discussed with respect to the supply of substrates and effectors of anion carriers and of key enzymes of the tricarboxylic acid cycle and fatty acid biosynthesis.     

Stable isotope peptide mass spectrometry to decipher amino acid metabolism in Dehalococcoides strain CBDB1

Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with (13)C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the α-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia.     

Factors regulating gluconeogenesis in chick embryo liver]

The ratio NAD+/NADH in cytoplasm and mitochondria of chicken embryo liver does not change up to the stage of hatching. After the hatching this ratio decreases 2-fold in both cytoplasm and mitochondria. The hatching is also accompanied by the decrease of total and mitochondrial contents of oxaloacetate and of oxaloacetate/malate ratio, the activity of citrate synthase and the ratio acetyl-CoA/CoA being unchanged.     

Engineered citrate synthase improves citramalic acid generation in Escherichia coli

The microbial product citramalic acid (citramalate) serves as a five-carbon precursor for the chemical synthesis of methacrylic acid. This biochemical is synthesized in Escherichia coli directly by the condensation of pyruvate and acetyl-CoA via the enzyme citramalate synthase. The principal competing enzyme with citramalate synthase is citrate synthase, which mediates the condensation reaction of oxaloacetate and acetyl-CoA to form citrate and begin the tricarboxylic acid cycle. A deletion in the gltA gene coding citrate synthase prevents acetyl-CoA flux into the tricarboxylic acid cycle, and thus necessitates the addition of glutamate. In this study the E. coli citrate synthase was engineered to contain point mutations intended to reduce the enzyme's affinity for acetyl-CoA, but not eliminate its activity. Cell growth, enzyme activity and citramalate production were compared in several variants in shake flasks and controlled fermenters. Citrate synthase GltA[F383M] not only facilitated cell growth without the presence of glutamate, but also improved the citramalate production by 125% compared with the control strain containing the native citrate synthase in batch fermentation. An exponential feeding strategy was employed in a fed-batch process using MEC626/pZE12-cimA harboring the GltA[F383M] variant, which generated over 60 g/L citramalate with a yield of 0.53 g citramalate/g glucose in 132 hr. These results demonstrate protein engineering can be used as an effective tool to redirect carbon flux by reducing enzyme activity and improve the microbial production of traditional commodity chemicals.     

The citrate cleavage pathway and lipogenesis in rat adipose tissue: replenishment of oxaloacetate

Fatty acid synthesis via the citrate cleavage pathway requires the continual replenishment of oxaloacetate within the mitochondria, probably by carboxylation of pyruvate. Malic enzyme, although present in adipose tissue, is completely localized in the cytoplasm and has insufficient activity to support lipogenesis. Pyruvate carboxylase was found to be active in both the mitochondria and cytoplasm of epididymal adipose tissue cells; it was dependent on both ATP and biotin. Alteractions in dietary conditions induced no significant changes in mitochondrial pyruvate carboxylase activity, but the soluble activity was depressed in fat-fed animals. The possible importance of the soluble activity in lipogenesis lies in its participation in a soluble malate transhydrogenation cycle with NAD malate dehydrogenase and malic enzyme, whereby a continual supply of NADPH is produced. Consequently, the pyruvate carboxylase in adipose tissue both generates mitochondrial oxaloacetate for the citrate cleavage pathway and supplies soluble NADPH for the conversion of acetyl-CoA to fatty acid.     

Characterization of citrate synthase from Geobacter sulfurreducens and evidence for a family of citrate synthases similar to those of eukaryotes throughout the Geobacteraceae

Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (kcat= 8.3 s(-1); Km= 14.1 and 4.3 microM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (Ki= 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K+ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.     

The kinetic properties of citrate synthase from rat liver mitochondria

1. Citrate synthase (EC 4.1.3.7) was purified 750-fold from rat liver. 2. Measurements of the Michaelis constants for the substrates of citrate synthase gave values of 16mum for acetyl-CoA and 2mum for oxaloacetate. Each value is independent of the concentration of the other substrate. 3. The inhibition of citrate synthase by ATP, ADP and AMP is competitive with respect to acetyl-CoA. With respect to oxaloacetate the inhibition by AMP is competitive, but the inhibition by ADP and ATP is mixed, being partially competitive. 4. At low concentrations of both substrates the inhibition by ATP is sigmoidal and a Hill plot exhibits a slope of 2.5. 5. The pH optimum of the enzyme is 8.7, and is not significantly affected by ATP. 6. Mg(2+) inhibits citrate synthase slightly, but relieves the inhibition caused by ATP in a complex manner. 7. At constant total adenine nucleotide concentration made up of various proportions of ATP, ADP and AMP, the activity of citrate synthase is governed by the concentration of the sum of the energy-rich phosphate bonds of ADP and ATP. 8. The sedimentation coefficient of the enzyme, as measured by activity sedimentation, is 6.3s, equivalent to molecular weight 95000.     

Identification of a Chlorobenzene Reductive Dehalogenase in Dehalococcoides sp. Strain CBDB1

A chlorobenzene reductive dehalogenase of the anaerobic dehalorespiring bacterium Dehalococcoides sp. strain CBDB1 was identified. Due to poor biomass yields, standard protein isolation procedures were not applicable. Therefore, cell extracts from cultures grown on trichlorobenzenes were separated by native polyacrylamide gel electrophoresis and analyzed directly for chlorobenzene reductive dehalogenase activity within gel fragments. Activity was found in a single band, even though electrophoretic separation was performed under aerobic conditions. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and nano-liquid chromatography-MALDI MS analysis of silver-stained replicas of the active band on native polyacrylamide gels identified a protein product of the cbdbA84 gene, now called cbrA. The cbdbA84 gene is one of 32 reductive dehalogenase homologous genes present in the genome of strain CBDB1. The chlorobenzene reductive dehalogenase identified in our study represents a member of the family of corrinoid/iron-sulfur cluster-containing reductive dehalogenases. No orthologs of cbdbA84 were found in the completely sequenced genomes of Dehalococcoides sp. strains 195 and BAV1 nor among the genes amplified from Dehalococcoides sp. strain FL2 or mixed cultures containing Dehalococcoides. Another dehalogenase homologue (cbdbA80) was expressed in cultures that contained 1,2,4-trichlorobenzene, but its role is unclear. Other highly expressed proteins identified with our approach included the major subunit of a protein annotated as formate dehydrogenase, transporter subunits, and a putative S-layer protein.     

Control of the tricarboxylate cycle and its interactions with glycolysis during acetate utilization in rat heart

1. Transient and steady-state changes caused by acetate utilization were studied in perfused rat heart. The transient period occupied 6min and steady-state changes were followed in a further 6min of perfusion. 2. In control perfusions glucose oxidation accounted for 75% of oxygen utilization; the remaining 25% was assumed to represent oxidation of glyceride fatty acids. With acetate in the steady state, acetate oxidation accounted for 80% of oxygen utilization, which increased by 20%; glucose oxidation was almost totally suppressed. The rate of tricarboxylate-cycle turnover increased by 67% with acetate perfusion. The net yield of ATP in the steady state was not altered by acetate. 3. Acetate oxidation increased muscle concentrations of acetyl-CoA, citrate, isocitrate, 2-oxoglutarate, glutamate, alanine, AMP and glucose 6-phosphate, and lowered those of CoA and aspartate; the concentrations of pyruvate, ATP and ADP showed no detectable change. The times for maximum changes were 1min, acetyl-CoA, CoA, alanine and AMP; 6min, citrate, isocitrate, glutamate and aspartate; 2-4min, 2-oxoglutarate. Malate concentration fell in the first minute and rose to a value somewhat greater than in the control by 6min. There was a transient and rapid rise in glucose 6-phosphate concentration in the first minute superimposed on the slower rise over 6min. 4. Acetate perfusion decreased the output of lactate, the muscle concentration of lactate and the [lactate]/[pyruvate] ratio in perfusion medium and muscle in the first minute; these returned to control values by 6min. 5. During the first minute acetate decreased oxygen consumption and lowered the net yield of ATP by 30% without any significant change in muscle ATP or ADP concentrations. 6. The specific radioactivities of cycle metabolites were measured during and after a 1min pulse of [1-(14)C]acetate delivered in the first and twelfth minutes of acetate perfusion. A model based on the known flow rates and concentrations of cycle metabolites was analysed by computer simulation. The model, which assumed single pools of cycle metabolites, fitted the data well with the inclusion of an isotope-exchange reaction between isocitrate and 2-oxoglutarate+bicarbonate. The exchange was verified by perfusions with [(14)C]bicarbonate. There was no evidence for isotope exchange between citrate and acetyl-CoA or between 2-oxoglutarate and malate. There was rapid isotope equilibration between 2-oxoglutarate and glutamate, but relatively poor isotope equilibration between malate and aspartate. 7. It is concluded that the citrate synthase reaction is displaced from equilibrium in rat heart, that isocitrate dehydrogenase and aconitate hydratase may approximate to equilibrium, that alanine aminotransferase is close to equilibrium, but that aspartate transamination is slow for reasons that have yet to be investigated. 8. The slow rise in citrate concentration as compared with the rapid rise in that of acetyl-CoA is attributed to the slow generation of oxaloacetate by aspartate aminotransferase. 9. It is proposed that the tricarboxylate cycle may operate as two spans: acetyl-CoA-->2-oxoglutarate, controlled by citrate synthase, and 2-oxoglutarate-->oxaloacetate, controlled by 2-oxoglutarate dehydrogenase; a scheme for cycle control during acetate oxidation is outlined. The initiating factors are considered to be changes in acetyl-CoA, CoA and AMP concentrations brought about by acetyl-CoA synthetase. 10. Evidence is presented for a transient inhibition of phosphofructokinase during the first minute of acetate perfusion that was not due to a rise in whole-tissue citrate concentration. The probable importance of metabolite compartmentation is stressed.     

Site-directed mutagenesis of citrate synthase; the role of the active-site aspartate in the binding of acetyl-CoA but not oxaloacetate

Asp-362, a potential key catalytic residue of Escherichia coli citrate synthase (citrate oxaloacetate-lyase [pro-3S)-CH2COO- ----acetyl-CoA), EC 4.1.3.7) has been converted to Gly-362 by oligonucleotide-directed mutagenesis. The mutant gene was completely sequenced, using a series of synthetic oligodeoxynucleotides spanning the structural gene to confirm that no additional mutations had occurred during genetic manipulation. The mutant gene was expressed in M13 bacteriophage and produced a protein which migrated in an identical manner to wild-type E. coli citrate synthase on SDS-polyacrylamide gels and which cross-reacted with E. coli citrate synthase antiserum. The mutant gene was subsequently recloned into pBR322 for large scale purification of the protein, and the resulting plasmid, pCS31, used to transform the citrate synthase deletion strain, W620. The mutant enzyme purified in an analogous manner to wild-type E. coli citrate synthase and expressed less than 2% of wild-type enzyme activity. The activity of the partial reactions catalysed by citrate synthase was similarly affected suggesting that this residual activity may be due to contaminating wild-type enzyme activity. The mutant citrate synthase retains a high-affinity NADH-binding site consistent with the protein preserving its overall structural integrity. Oxaloacetate binding to the protein is unaffected by the Asp-362 to Gly-362 mutation. Binding of the acetyl-CoA analogue, carboxymethyl-CoA, could not be detected in the mutant protein indicating that the lack of catalytic competence is due primarily to the inability of the protein to bind the second substrate, acetyl-CoA.     

Substrate-inhibiton by acetyl-CoA in the condensation reaction between oxaloacetate and acetyl-CoA catalyzed by citrate synthase from pig heart

Deviations from Michealis-Menten kinetics in the pig-heart citrate synthase (citrate-oxaloacetate-lyase(pro-3S-CH2-COO-leads to acetyl-CoA), EC 4.1.3.7) system have been characterized and analyzed in view of the kinetic theory described in the preceding paper. The enzymic condensation reaction between acetyl-CoA and oxaloacetate is subject to substrate-inhibition by acetyl-CoA. This can be attributed to the formation of a productive enzyme-acetyl-CoA complex with a dissociation constant of 110 uM. The binding of acetyl-CoA to the enzyme decreases the on-velocity constant for oxaloacetate-binding from 4000 min-1- micrometer-1 to 1700 min-1-micrometer-1. The affinity of citrate synthase for oxaloacetate increase at least 20-fold on the binding of acetyl-CoA. The latter cooperativity effect can be attributed to a more than 45-fold decrease of the off-velocity constant for oxaloacetate-binding.     

In vitro mutagenesis of Escherichia coli citrate synthase to clarify the locations of ligand binding sites

In vitro mutagenesis techniques have been used to investigate two structure-function questions relating to the allosteric citrate synthase of Escherichia coli. The first question concerns the binding site of alpha-keto-glutarate, which is a structural analogue of the substrate oxaloacetate and yet has been suggested to be an allosteric inhibitor of the enzyme. Using oligonucleotide-directed mutagenesis of the cloned E. coli citrate synthase gene, we prepared missense mutants, designated CS226H----Q and CS229H----Q, in which histidine residues at positions 226 and 229, respectively, were replaced by glutamine. In the homologous pig heart citrate synthase it is known (Wiegand, G., and Remington, S. J. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 97-117) that the equivalent of His-229 helps to bind oxaloacetate, while the equivalent of His-226 is nearby. Kinetic and ligand binding measurements showed that CS226H----Q had a reduced affinity for oxaloacetate and alpha-ketoglutarate, while CS229H----Q bound oxaloacetate even less effectively, and was not inhibited by alpha-ketoglutarate at all under our conditions. This parallel loss of binding affinities for oxaloacetate and alpha-ketoglutarate, in two mutants altered in residues at the active site of E. coli citrate synthase, strongly suggests that inhibition of this enzyme by alpha-ketoglutarate is not allosteric but occurs by competitive inhibition at the active site. The second question investigated was whether the known inhibition by acetyl-CoA of binding of NADH, an allosteric inhibitor of E. coli citrate synthase, occurs heterotropically, as an indirect result of acetyl-CoA binding at the active site, or directly, by competition at the allosteric NADH binding site. Using existing restriction sites in the cloned E. coli citrate synthase gene, we prepared a deletion mutant which lacked 24 amino acids near what is predicted to the acetyl-CoA-binding portion of the active site. The mutant protein was inactive, and acetyl-CoA did not bind to the active site but still inhibited NADH binding. Thus acetyl-CoA can interact with both the allosteric and the active sites of this enzyme.     

Aspects of ketogenesis: Control and mechanism of ketone-body formation in isolated RAT-liver mitochondria

The synthesis of ketone bodies by intact isolated rat-liver mitochondria has been studied at varying rates of acetyl-CoA production and of acetyl-CoA utilization in the Krebs cycle. Factors which enhanced the rate of acetyl-CoA production caused an increase in the fraction of acetyl-CoA which was incorporated into ketone bodies. On the other hand, it was found that factors which stimulated the formation of citrate lowered the relative rate of ketogenesis. It is concluded that acetyl-CoA is preferentially used for citrate synthesis, if the level of oxaloacetate in the mitochondrial matrix space is adequate. The intramitochondrial level of oxaloacetate, which is determined by the malate concentration and the ratio of NADH over NAD+, is the main factor controlling the rate of citrate synthesis. The ATP/ADP ratio per se does not affect the activity of citrate synthase in this in vitro system. Ketogenesis can be described as an overflow of acetyl-groups: Ketone-body formation is stimulated only when the rate of acetyl-CoA production increases beyond the capacity for citrate synthesis. The interaction between fatty acid oxidation and pyruvate metabolism and the effects of long-chain acyl-CoA on mitochondrial metabolism are discussed. Ketone bodies which were generated during the oxidation of [1-14C] fatty acids were preferentially labelled in their carboxyl group. This carboxyl group had the same specific activity as the acetyl-CoA pool, whereas the specific activity of the acetone moiety of acetoacetate was much lower, especially at low rates of ketone-body formation. The activities of acetoacetyl-CoA deacylase and the hydroxymethylglutaryl-CoA (HMG-CoA) pathway were compared in soluble and mitochondrial fractions of rat- and cow-liver in different ketotic states. In rat-liver mitochondria, both pathways of acetoacetate synthesis were stimulated upon starvation or in alloxan diabetes. In cow liver, only the HMG-CoA pathway was increased during ketosis in the mitochondrial as well as in the soluble fraction.     

Poly-β-hydroxybutyrate (PHB) biosynthesis,tricarboxylic acid activity and PHB content in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) root nodule

An experiment was conducted to assess the relationship between poly-β-hydroxybutyrate (PHB) biosynthesis and tricarboxylic acid (TCA) activity in desi and kabuli chickpea (Cicer arietinum L.) genotypes. The specific activities of enzymes of PHB metabolism viz., β-ketothiolase (PHB-A), acetoacetyl coenzyme A reductase (PHB-B) and PHB synthase (PHB-C), and those of tricarboxylic acid cycle (citrate synthase (CS) and malate dehydrogenase (MDH) under symbiosis were measured in bacteroids and compared with the PHB accumulation in the nodule and the root. The significant positive correlation was observed between shoot and nodule mass and PHB-A, PHB-B, and PHB-C activities. However, nodule and shoot weights were not significantly correlated with PHB content either in the roots or nodules. The same was true for PHB levels and citrate synthase activity. MDH activity showed a significant negative correlation with nodule PHB. A marked variation and an age dependant increase in malate dehydrogenase activity were measured. A higher capacity for malate oxidation by an increased MDH is likely alter the balance between malate decarboxylation and oxidation, resulting in a higher steady-state concentration of oxaloacetate and that may favor the utilization of acetyl-CoA in the TCA cycle rather than for the synthesis of PHB.     

Inhibitors of metabolic reactions. Scope and limitation of acyl-CoA-analogue CoA-thioethers.

Substrate and intermediate analogue inhibitors of enzymes were prepared in which the thioester oxygen of acyl-CoA substrates is replaced by hydrogen with formation of CoA-thioethers. Experiments performed with ATP citrate lyase and S-(3,4-dicarboxy-3-hydroxybutyl)-CoA are consistent with citryl-CoA but not with citryl-enzyme being the direct precursor of the products acetyl-CoA and oxaloacetate. Consistent with these results, a previously described isotopic exchange between acetyl-CoA and [3H]CoASH, indicating the formation of an acetyl-enzyme in the reaction pathway, could not be confirmed. Substrate analogue CoA-thioethers of malate synthase are inhibitors endowed with the affinity of the substrates. Acetyl carboxylase and fatty acid synthetase are not inhibited by the substrate analogue S-ethyl-CoA; S-carboxyethyl-CoA, which could substitute for malonyl-CoA, is likewise not inhibitory. An explanation is proposed. Previously suggested roles of S-carboxymethyl-CoA, an acetyl-CoA-related inhibitor of citrate synthase, are discussed in the light of new experimental data. S-Acetyl, S-propionyl and S-carboxymethyl derivatives of 1,N6-etheno-CoA loose the high affinity of their CoA-counterparts to citrate synthase, probably because the ethylene group prevents proper binding to the enzyme.     

Changes in the contents of adenine nucleotides and intermediates of glycolysis and citric acid cycle in flight muscle of the locust upon flight and their relationship to the control of the cycle

1. The contents of some intermediates of glycolysis, the citric acid cycle and adenine nucleotides have been measured in the freeze-clamped locust flight muscle at rest and after 10s and 3min flight. The contents of glucose 6-phosphate, pyruvate, alanine and especially fructose bisphosphate and triose phosphates increased markedly upon flight. The content of acetyl-CoA is decreased after 3min flight whereas that of acetylcarnitine is decreased markedly after 10s flight, but returns towards the resting value after 3min flight. The content of citrate is markedly decreased after both 10s and 3min flight, whereas that of isocitrate is changed very little after 10s and is increased by 50% after 3min. The content of oxaloacetate is very low in insect flight muscle and hence it was measured by a sensitive radiochemical assay. The content of oxaloacetate increased about 2-fold after 3min flight. A similar change was observed in the content of malate. The content of ATP decreased about 15%, whereas those of ADP and AMP increased about 2-fold after 3min flight. 2. Calculations based on O(2) uptake of the intact insect indicate that the rate of the citric acid cycle must be increased >100-fold during flight. Consequently, if citrate synthase catalyses a non-equilibrium reaction, the activity of the enzyme must increase >100-fold during flight. However, changes in the concentrations of possible regulators of citrate synthase, oxaloacetate, acetyl-CoA and citrate (which is an allosteric inhibitor), are not sufficient to account for this change in activity. It is concluded that there may be much larger changes in the free concentration of oxaloacetate than are indicated by the changes in the total content of this metabolite or that other unknown factors must play an additional role in the regulation of citrate synthase activity. 3. The increased content of oxaloacetate could be produced via pyruvate carboxylase, which may be stimulated during the early stages of flight by the increased concentration of pyruvate. 4. The decreases in the concentrations of citrate and alpha-oxoglutarate indicate that isocitrate dehydrogenase and oxoglutarate dehydrogenase may be stimulated by factors other than their pathway substrates during the early stages of flight. 5. Calculated mitochondrial and cytosolic NAD(+)/NADH ratios are both increased upon flight. The change in the mitochondrial ratio indicates the importance of the intramitochondrial ATP/ADP concentration ratio in the regulation of the rate of electron transfer in this muscle.     

Metabolic network analysis of Penicillium chrysogenum using (13)C-labeled glucose

Using (13)C-labeled glucose fed to a penicillin-overproducing strain of Penicillium chrysogenum, the intracellular fluxes were quantified, and the presence of two new pathways, not previously described in this organism, is suggested. Thus, glycine was synthesized not only by serine hydroxymethyltransferase, but also by threonine aldolase. The formation of cytosolic acetyl-CoA was found to be synthesized both via the citrate lyase-catalyzed reaction and by degradation of the penicillin side-chain precursor, phenoxyacetic acid. Furthermore, the experimental data indicate that the main activities of homocitrate synthase and alpha-isopropylmalate synthase are located in the cytosol. All experimental data on the labeling patterns were obtained using gas chromatography-mass spectrometry, which is faster and more sensitive than the nuclear magnetic resonance methods usually applied for analysis of labeling patterns.     

Genome analysis and heterologous expression of acetate-activating enzymes in the anammox bacterium Kuenenia stuttgartiensis

Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene (acs) of Kuenenia stuttgartiensis, encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs, the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria.