Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.3.1 (citrate synthase)
 4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The synthesis of a new coenzyme A analogue, N6-[N-(6-aminohexyl)carbamoylmethyl]-CoA, suitable for immobilisation through its terminal amino group to support matrices, is described. The synthetic route starts with bis(CoA) and involves the following steps: alkylation with iodoacetic acid and rearrangement yielding bis(N6-carboxymethyl-CoA), elongation of the carboxymethyl terminal with 1,6-diaminohexane using carbodiimide to yield bis(N6-[N-(6-aminohexyl)-carbamoylmethyl]-CoA) and finally the splitting of this bis[CoA analogue) through reduction with dithiothreitol to give the final product in approximately 10% overall yield. This CoA analogue showed 'coenzymic activity' with the enzymes acetyl-CoA synthetase, phosphotransacetylase and succinic thiokinase. Covalent binding of the CoA analogue to Sepharose 4B was normally carried out using its S-(5-thio-2-nitrobenzoic acid) derivative as this allows a convenient way for determining the amount of ligand coupled, based on the amount of 5-thio-2-nitrobenzoic acid liberated from the gel after reduction with dithiothreitol. After covalent binding of the CoA analogue to water-soluble activated dextran 70, the analogue was recycled while present in an ultrafiltration cell using the enzymes phosphotransacetylase and citrate synthase. The reaction was followed by measuring the citrate formed on addition of acetylphosphate and oxaloacetate. In affinity chromatographic studies it was shown that the CoA-Sepharose preparation could bind the CoA-dependent enzymes citrate synthase and succinic thiokinase and these could be biospecifically eluted using soluble CoA.
 ...
PMID:N6-[N-(6-Aminohexyl)carbamoylmethyl]-coenzyme A. Synthesis and application in affinity chromatography and as an immobilized active coenzyme. 57 88

Carnitine acetyltransferase was isolated from yeast Saccharomyces cerevisiae with an apparent molecular weight of 400,000. The enzyme contains identical subunits of 65,000 Da. The Km values of the isolated enzyme for acetyl-CoA and for carnitine were 17.7 microM and 180 microM, respectively. Carnitine acetyltransferase is an inducible enzyme, a 15-fold increase in the enzyme activity was found when the cells were grown on glycerol instead of glucose. Carnitine acetyltransferase, similarly to citrate synthase, has a double localization (approx. 80% of the enzyme is mitochondrial), while acetyl-CoA synthetase was found only in the cytosol. In the mitochondria carnitine acetyltransferase is located in the matrix space. The incorporation of 14C into CO2 and in lipids showed a similar ratio, 2.9 and 2.6, when the substrate was [1-14C]acetate and [1-14C]acetylcarnitine, respectively. Based on these results carnitine acetyltransferase can be considered as an enzyme necessary for acetate metabolism by transporting the activated acetyl group from the cytosol into the mitochondrial matrix.
 ...
PMID:Isolation and characterization of carnitine acetyltransferase from S. cerevisiae. 189 91

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.
 ...
PMID:The intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm. 439 82

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.
 ...
PMID:Control of the tricarboxylate cycle and its interactions with glycolysis during acetate utilization in rat heart. 544 22

Cells of the aerotolerant anaerobe Giardia lamblia respire in the presence of oxygen. Endogenous respiration is stimulated by glucose but not by other carbohydrates and Krebs cycle intermediates. Endogenous and glucose-stimulated respiration are insensitive to cyanide, malonate, and 2,4-dinitrophenol, but are inhibited by atabrin and iodoacetamide. G. lamblia produces ethanol, acetate and CO2 both aerobically and anaerobically either from endogenous reserves or exogenous glucose. Molecular hydrogen is not produced. The following enzyme activities were detected in homogenates: hexokinase, fructose-biphosphate aldolase, pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malate dehydrogenase (decarboxylating), pyruvate synthase, acetyl-CoA synthetase, alcohol dehydrogenase (NADP+), NADH dehydrogenase, NADPH dehydrogenase, NADPH oxidoreductase and superoxide dismutase. The enzymes of energy and carbohydrate metabolism are nonsedimentable (109 000 x g for 30 min). Activities of lactate dehydrogenase, hydrogenase, phosphate acetyltransferase, acetate kinase, citrate synthase, succinate dehydrogenase, fumarate hydratase and catalase were below the limits of detection. The results suggest the occurrence of glycolysis, energy production by substrate level phosphorylation and a flavin, iron-sulfur protein mediated electron transport system as well as the absence of cytochrome mediated oxidative phosphorylation and functional Krebs cycle.
 ...
PMID:Energy metabolism of the anaerobic protozoon Giardia lamblia. 610 7

The activities of five enzymes involved in acetyl-CoA synthesis, pyruvate dehydrogenase complex, ATP citrate lyase, carnitine acetyltransferase, acetyl-CoA synthetase, and citrate synthase, were determined in normal nucleus interpeduncularis and nucleus interpeduncularis in which cholinergic terminals were removed following lesion of the habenulointerpeduncular tract. The activities of aspartate transaminase, fumarase, and GABA transaminase also were determined to compare the effect of lesion on other mitochondrial enzymes which are not linked to the biosynthesis of ACh. In normal nucleus interpeduncularis the activities of carnitine acetyltransferase and pyruvate dehydrogenase complex were higher than the activity of ChAT (choline acetyltransferase), whereas the activities of acetyl-CoA synthetase and citrate synthase were considerably lower than that of ChAT. The effect of the lesion separated the enzymes into two groups: the activities of pyruvate dehydrogenase complex, carnitine acetyltransferase, fumarase and aspartate transaminase decreased by 30--40%, whereas the activities of the other enzymes descreased 5--15%. ChAT activity was in all cases less than 15% of normal. It could be concluded that none of the acetyl-CoA synthesizing enzymes decreased to the degree that ChAT did. Only pyruvate dehydrogenase complex and carnitine acetyltransferase seem to be localized in cholinergic terminals to a significant degree. ATP citrate lyase as well as acetyl-CoA synthetase seem to have less significance in supporting acetyl-CoA formation in cholinergic nerve terminals.
 ...
PMID:Acetyl-CoA synthesizing enzymes in cholinergic nerve terminals. 610 88

The activities of choline acetyltransferase and ATP-citrate lyase were significantly correlated (r = 0.995) in fractions of small and large synaptosomes isolated from rat hippocampus and cerebellum. The activities of these two enzymes did not correlate with those of pyruvate dehydrogenase, carnitine acetyltransferase, citrate synthase, acetyl-CoA synthetase, lactate dehydrogenase, or with the rate of high-affinity glutamate uptake in the synaptosomal fractions. The results provide additional evidence linking ATP-citrate lyase to the cholinergic system in the brain.
 ...
PMID:ATP-citrate lyase and other enzymes of acetyl-CoA metabolism in fractions of small and large synaptosomes from rat brain hippocampus and cerebellum. 613 19

Two nuclear genes, RTG1 and RTG2, which sense the functional state of yeast mitochondria, have been described recently. Yeast strains with null alleles of either of these two genes (delta rtg1, delta rtg2) cannot grow on acetate as the sole carbon source and are auxotrophic for glutamate and aspartate. We report here a series of metabolic experiments and enzyme activity measurements that were made in an attempt to determine the reason for the acetate- phenotype and the glutamate/aspartate auxotrophy. Decreases in the activities (approximately 50%) in mitochondrial citrate synthase (CS1), acetyl-CoA synthetase, NAD isocitrate dehydrogenase, and pyruvate carboxylase were noted. When CS1 was overexpressed in the delta rtg1 and delta rtg2 mutants, these strains could grow on acetate but were still auxotrophic for glutamate/aspartate. We propose that, in the mutant strain, CS1 activity becomes limiting for efficient acetate utilization, but that other complex metabolic interactions are affected, limiting production of intermediates that would allow synthesis of glutamic and aspartic acids.
 ...
PMID:Enzymatic and metabolic studies on retrograde regulation mutants of yeast. 772 18

Corynebacterium glutamicum lacking the succinate dehydrogenase complex can produce succinate aerobically with acetate representing the major byproduct. Efforts to increase succinate production involved deletion of acetate formation pathways and overexpression of anaplerotic pathways, but acetate formation could not be completely eliminated. To address this issue, we constructed a pathway for recycling wasted carbon in succinate-producing C. glutamicum. The acetyl-CoA synthetase from Bacillus subtilis was heterologously introduced into C. glutamicum for the first time. The engineered strain ZX1 (pEacsA) did not secrete acetate and produced succinate with a yield of 0.50 mol (mol glucose)(-1). Moreover, in order to drive more carbon towards succinate biosynthesis, the native citrate synthase encoded by gltA was overexpressed, leading to strain ZX1 (pEacsAgltA), which showed a 22% increase in succinate yield and a 62% decrease in pyruvate yield compared to strain ZX1 (pEacsA). In fed-batch cultivations, strain ZX1 (pEacsAgltA) produced 241 mM succinate with an average volumetric productivity of 3.55 mM h(-1) and an average yield of 0.63 mol (mol glucose) (-1), making it a promising platform for the aerobic production of succinate at large scale.
 ...
PMID:Engineering of acetate recycling and citrate synthase to improve aerobic succinate production in Corynebacterium glutamicum. 2359 75

Recently, butanols (1-butanol, 2-butanol and iso-butanol) have generated attention as alternative gasoline additives. Butanols have several properties favorable in comparison to ethanol, and strong interest therefore exists in the reconstruction of the 1-butanol pathway in commonly used industrial microorganisms. In the present study, the biosynthetic pathway for 1-butanol production was reconstructed in the yeast Saccharomyces cerevisiae. In addition to introducing heterologous enzymes for butanol production, we engineered yeast to have increased flux toward cytosolic acetyl-CoA, the precursor metabolite for 1-butanol biosynthesis. This was done through introduction of a plasmid-containing genes for alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA synthetase (ACS), and acetyl-CoA acetyltransferase (ERG10), as well as the use of strains containing deletions in the malate synthase (MLS1) or citrate synthase (CIT2) genes. Our results show a trend to increased butanol production in strains engineered for increased cytosolic acetyl-CoA levels, with the best-producing strains having maximal butanol titers of 16.3 mg/l. This represents a 6.5-fold improvement in butanol titers compared to previous values reported for yeast and demonstrates the importance of an improved cytosolic acetyl-CoA supply for heterologous butanol production by this organism.
 ...
PMID:Improving biobutanol production in engineered Saccharomyces cerevisiae by manipulation of acetyl-CoA metabolism. 2376 Apr 99


1 2 Next >>