Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.3.1 (citrate synthase)
4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The absolute separation of the four stereoisomeric configurations of methylcitric acid can be achieved on a nonchiral stationary phase SE30 capillary column using the corresponding O-acetylated (tri-(-)-2-butyl ester derivatives. Identification of the separated isomers was done using methylcitric acid produced by si-citrate synthase and methylcitrate synthase of Candida lipolitica. si-Citrate synthase produces the (2S,3S)-, (2S,3R)- and a small amount of the (2R,3S)-isomers. Methylcitrate synthase produces the (2R,3S)-isomer, indicating that this enzyme is more stereospecific than the animal citrate synthase enzyme. The (2R,3R)-isomer may act as an inhibitor of aconitase.
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PMID:Identification of the stereoisomeric configurations of methylcitric acid produced by si-citrate synthase and methylcitrate synthase using capillary gas chromatography-mass spectrometry. 770 98

A multienzyme complex of tricarboxylic acid cycle enzymes, catalysing the consecutive reactions from fumarate to 2-oxoglutarate, has been identified in extracts of Pseudomonas aeruginosa prepared by gentle osmotic lysis of the cells. The individual enzyme activities of fumarase, malate dehydrogenase, citrate synthase, aconitase and isocitrate dehydrogenase can be used to reconstitute the complex. The citrate synthase isoenzymes, CSI and CSII, from this organism can be used either together or as the individual activities to reconstitute the complex. No complex can be reformed in the absence of CSI or CSII. Which CS isoenzyme predominates in the complex depends on the phase of growth at which the cells were harvested and the extract prepared. More CSI was found in the complex during exponential growth, whereas CSII predominated during the stationary phase. The results support the idea of a 'metabolon' in this organism, with the composition of the CS component varying during the growth cycle.
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PMID:Identification of a multienzyme complex of the tricarboxylic acid cycle enzymes containing citrate synthase isoenzymes from Pseudomonas aeruginosa. 861 Nov 53

In work previously reported (J. A. Gutierrez, P. J. Crowley, D. P. Brown, J. D. Hillman, P. Youngman, and A. S. Bleiweis, J. Bacteriol. 178:4166-4175, 1996), a Tn917 transposon-generated mutant of Streptococcus mutans JH1005 unable to synthesize glutamate anaerobically was isolated and the insertion point of the transposon was determined to be in the icd gene encoding isocitrate dehydrogenase (ICDH). The intact icd gene of S. mutans has now been isolated from an S. mutans genomic plasmid library by complementation of an icd mutation in Escherichia coli host strain EB106. Genetic analysis of the complementing plasmid pJG400 revealed an open reading frame (ORF) of 1,182 nucleotides which encoded an enzyme of 393 amino acids with a predicted molecular mass of 43 kDa. The nucleotide sequence contained regions of high (60 to 72%) homology with icd genes from three other bacterial species. Immediately 5' of the icd gene, we discovered an ORF of 1,119 nucleotides in length, designated citZ, encoding a homolog of known citrate synthase genes from other bacteria. This ORF encoded a predicted protein of 372 amino acids with a molecular mass of 43 kDa. Furthermore, plasmid pJG400 was also able to complement a citrate synthase (gltA) mutation of E. coli W620. The enzyme activities of both ICDH, found to be NAD+ dependent, and citrate synthase were measured in cell extracts of wild-type S. mutans and E. coli mutants harboring plasmid pJG400. The region 5' from the citZ gene also revealed a partial ORF encoding 264 carboxy-terminal amino acids of a putative aconitase gene. The genetic and biochemical evidence indicates that S. mutans possesses the enzymes required to convert acetyl coenzyme A and oxalacetate to alpha-ketoglutarate, which is necessary for the synthesis of glutamic acid. Indeed, S. mutans JH1005 was shown to assimilate ammonia as a sole source of nitrogen in minimal medium devoid of organic nitrogen sources.
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PMID:Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans. 900 16

Escherichia coli contains two genes (acnA and acnB) encoding aconitase activities. An acnB mutant was engineered by replacing the chromosomal acnB gene by an internally deleted derivative containing a tetR cassette. An acnB double mutant was then made by transducing a previously constructed acnA::kanR mutation into the acnB::tetR strain. Western blotting confirmed that the AcnA and AcnB proteins were no longer produced by the corresponding mutants and PCR analysis showed that the chromosomal acnB gene had been replaced by the disrupted gene. Aerobic and anaerobic growth in glucose minimal medium were impaired but not abolished by the acnB mutation, indicating that the lesion is partially complemented by the acnA+ gene, and growth was enhanced by glutamate. The acnAB double mutant would not grow on unsupplemented glucose minimal medium and although it responded to glutamate like a typical auxotroph under anaerobic conditions, under aerobic conditions no response to glutamate was observed before it was over-grown by 'revertants' lacking citrate synthase (acnAB gltA). The acnAB double mutant retained a low but significant aconitase activity (< or = 5% of wild-type), designated AcnC. Enzymological and regulatory studies with acn-lacZ fusions indicated that AcnB is the major aconitase, which is synthesized earlier in the growth cycle than AcnA, and subject to catabolite and anaerobic repression.
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PMID:Construction and properties of aconitase mutants of Escherichia coli. 920 58

The enzymes which are responsible for catalyzing sequential reactions in several metabolic pathways have been proposed to be highly organized in supramolecular complexes termed metabolons. However, the in situ existence of these weak complexes is difficult to demonstrate because many of them are dissociated during isolation due to dilution effects. Consequently, the metabolon concept is subject to controversy. A model system consisting of genetically prepared bienzymatic fusion proteins has been used to immobilize sequential metabolic enzymes in close proximity and to demonstrate possible kinetic advantages of metabolons. These experiments use the sequential Krebs TCA cycle enzymes from yeast mitochondrial malate dehydrogenase (MDH), citrate synthase (CS), and aconitase (ACO). Using the porcine high-definition structures of these three enzymes, we have performed computer-modeling studies in order to understand how the molecules may interact. Among the thousands of docking orientations we have tried, one was found to respond to the structural and experimental constraints from the results obtained with the yeast fusion proteins. Interestingly, this quinary structure model shows substantial interacting surface areas with spatial and electrostatic complementarities which make the complex thermodynamically stable. This structure also contains an unbroken electrostatically favorable channel connecting the active sites of ACO and CS, as well as the one previously reported between CS and MDH active sites. Charged amino acids which could be involved in interactions stabilizing the complex have been identified. This model will be used as the basis for further experimental work on the structure of the Krebs TCA cycle metabolon.
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PMID:Model of a quinary structure between Krebs TCA cycle enzymes: a model for the metabolon. 940 Mar 65

A study was undertaken to estimate the activities of the key enzymes of glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle in purified rat spermatocytes and spermatids, which have been shown to die in glucose-containing medium and require lactate/pyruvate for maintaining normal ATP concentrations. The aim was to elucidate the changes in the glycolytic and oxidative potential of germ cells undergoing meiosis. Pachytene spermatocytes and round spermatids from adult rat testis were purified to approximately 90% purity by trypsin digestion followed by a combination of centrifugal elutriation and Percoll density gradient centrifugation. After the purity and viability of these cells had been established, their contents of hexokinase, phosphofructokinase, lactate dehydrogenase (LDH) and LDH-X of glycolysis, glucose 6-phosphate dehydrogenase of the pentose phosphate pathway and citrate synthase, aconitase, malate dehydrogenase and 2-oxoglutarate dehydrogenase of the TCA cycle were estimated. These enzymes were also estimated in epididymal spermatozoa for comparison with the testicular germ cells. The results indicate greater activity of glycolytic and pentose phosphate pathway enzymes in spermatocytes than in spermatids, which exhibited greater activity of TCA cycle enzymes than the former. The difference in activity was statistically significant for most of the enzymes studied. In contrast, spermatozoa exhibited markedly greater activity of glycolytic enzymes and significantly lower activity of pentose phosphate pathway and TCA cycle enzymes than did the testicular germ cells. We conclude that the unusual dependence of spermatids exclusively on lactate may be due to their lower glycolytic potential, whereas spermatocytes with comparatively greater glycolytic activity have an intermediate dependence on lactate and are therefore able to utilise lactate, pyruvate, or both, while retaining a better ability to utilise glucose. Spermatozoa with the greatest glycolytic potential and the lowest TCA cycle activity appear to be 'programmed' to utilise exclusively glucose/fructose for energy.
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PMID:Changes in carbohydrate metabolism of testicular germ cells during meiosis in the rat. 953 8

Strips of rat soleus muscle were incubated in media containing a superoxide generating system and/or the nitric oxide donor sodium nitroprusside (SNP) before the maximal catalytic activities of aconitase, citrate synthase, and oxoglutarate dehydrogenase were measured. The maximal activities of aconitase and oxoglutarate dehydrogenase were both decreased by 25-30% by superoxide anions; however, only the maximal activity of aconitase was decreased, by approximately 50%, by incubation of muscles with SNP. Furthermore, when both superoxide and NO were present in the medium, aconitase activity was decreased by 70%. The maximal activity of citrate synthase was not affected by any of the treatments. This is the first time that superoxide anions or NO has been shown to inactivate aconitase and oxoglutarate dehydrogenase in skeletal muscle. It is suggested that these effects may be responsible for some alterations in skeletal muscle metabolism, and these possibilities are discussed.
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PMID:Inactivation of aconitase and oxoglutarate dehydrogenase in skeletal muscle in vitro by superoxide anions and/or nitric oxide. 971 27

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.
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PMID:The tricarboxylic acid cycle of Helicobacter pylori. 1009 6

Iron modulates the expression of the critical citric acid cycle enzyme aconitase via a translational mechanism involving iron regulatory proteins. Thus, the present study was undertaken to investigate the consequences of iron perturbation on citric acid cycle activity, oxidative phosphorylation and mitochondrial respiration in the human cell line K-562. In agreement with previous data iron increases the activity of mitochondrial aconitase while it is reduced upon addition of the iron chelator desferrioxamine (DFO). Interestingly, iron also positively affects three other citric acid cycle enzymes, namely citrate synthase, isocitric dehydrogenase, and succinate dehydrogenase, while DFO decreases the activity of these enzymes. Consequently, iron supplementation results in increased formation of reducing equivalents (NADH) by the citric acid cycle, and thus in increased mitochondrial oxygen consumption and ATP formation via oxidative phosphorylation as shown herein. This in turn leads to downregulation of glucose utilization. In contrast, all these metabolic pathways are reduced upon iron depletion, and thus glycolysis and lactate formation are significantly increased in order to compensate for the decrease in ATP production via oxidative phosphorylation in the presence of DFO. Our results point to a complex interaction between iron homeostasis, oxygen supply and cellular energy metabolism in human cells.
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PMID:Iron-dependent changes in cellular energy metabolism: influence on citric acid cycle and oxidative phosphorylation. 1055 22

Friedreich's ataxia (FRDA) is an autosomal recessive disorder with a frequency of 1 in 50 000 live births. In 97% of patients it is caused by the abnormal expansion of a GAA repeat in intron 1 of the FRDA gene on chromosome 9, which encodes a 210 amino acid protein called frataxin. Frataxin is widely expressed and has been localized to mitochondria although its function is unknown. We have investigated mitochondrial function, mitochondrial DNA levels, aconitase activity and iron content in tissues from FRDA patients. There were significant reductions in the activities of complex I, complex II/III and aconitase in FRDA heart. Respiratory chain and aconitase activities were decreased although not significantly in skeletal muscle, but were normal in FRDA cerebellum and dorsal root ganglia, although there was a mild decrease in aconitase activity in the latter. Mitochondrial DNA levels were reduced in FRDA heart and skeletal muscle, although in skeletal muscle this was paralleled by a decline in citrate synthase activity. Increased iron deposition was seen in FRDA heart, liver and spleen in a pattern consistent with a mitochondrial location. The iron accumulation, mitochondrial respiratory chain and aconitase dysfunction and mitochondrial DNA depletion in FRDA heart samples largely paralleled those in the yeast YFH1 knockout model, suggesting that frataxin may be involved in mitochondrial iron regulation or iron sulphur centre synthesis. However, the severe deficiency in aconitase activity also suggests that oxidant stress may induce a self-amplifying cycle of oxidative damage and mitochondrial dysfunction, which may contribute to cellular toxicity.
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PMID:Clinical, biochemical and molecular genetic correlations in Friedreich's ataxia. 1060 38


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