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)

Citrate synthase activity of Saccharomyces cerevisiae was determined by a radioactive assay procedure and the reaction product, 14C-citric acid, was identified by chromatographic techniques. ATP, d-ATP, GTP and NADH were most inhibitory to the citrate synthase invitro. The activity was inhibited to a lesser extent by ADP, UTP, and NADP whereas, AMP and CTP were much less inhibitory. NADH, like NAD, glutamic acid, glutamine, arginine, ornithine, proline, aspartic acid and alpha-ketoglutarate exhibited no inhibition. These results have been discussed in the light of the role of citrate synthase for the energy metabolism and glutamic acid biosynthesis.
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PMID:Regulation of citrate synthase activity of Saccharomyces cerevisiae. 0

Relationship of citrate synthase (EC 4.1.3.7) to the biosynthesis of glutamic acid was investigated by characterizing a new glutamic acid auxotroph FL100-D1 (glu 3) of Saccharomyces cerevisiae. Nutritional requirement of the mutant was satisfied by L-glutamic acid, L-glutamic acid peptide as well as several analogs of glutamic acid, but not by proline, ornithine, arginine, lysine or aspartic acid. The mutant was unable to utilize nonfermentable carbon sources, glycerol, acetate or lactate. Mutant glu3 unlike aconitaseless glutamic acid auxotroph glu 1, failed to accumulate 14C-citric acid in vivo from 1-14C-sodium acetate or U-14C-glutamic acid. Both spectrophotometric and radioactive assay procedures demonstrated a lack of significant citrate synthase activity in the dialysed extract of the mutant compared to the wild type strain. Mutant glu 3 complemented with glu 1 and glu 2 individually in vivo and exhibited a significant aconitase (EC 4.2.1.3) activity in vitro.
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PMID:Citrate synthaseless glutamic acid auxotroph of Saccharomyces cerevisiae. 110 43

The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp----Asn and Asp----Gly mutations. The Asp----Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563].
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PMID:Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid. 168 5

The crystal structure of the ternary complex citrate synthase-oxaloacetate-carboxymethyl coenzyme A has been solved to a resolution of 1.9 A and refined to a conventional crystallographic R factor of 0.185. The structure resembles a proposed transition state of the condensation reaction and suggests that the condensation reaction proceeds through a neutral enol rather than an enolate intermediate. A mechanism for the condensation reaction is proposed which involves the participation of three key catalytic groups (Asp 375, His 274, and His 320) in two distinct steps. The proposed mechanism invokes concerted general acid-base catalysis twice to explain both the energetics of the reaction and the experimentally observed inversion of stereochemistry at the attacking carbon atom.
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PMID:Proposed mechanism for the condensation reaction of citrate synthase: 1.9-A structure of the ternary complex with oxaloacetate and carboxymethyl coenzyme A. 233

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.
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PMID:Site-directed mutagenesis of citrate synthase; the role of the active-site aspartate in the binding of acetyl-CoA but not oxaloacetate. 328 13

During exponential growth, ordinary colorless (OC) plants of Blastocladiella emersonii consumed little glucose and produced no lactic acid. Similarly, resistant sporangial (RS) plants did not utilize glucose or produce lactic acid during the first 24 hr of exponential growth. During the next 24 hr of RS development, glucose was consumed with the concomitant production of lactic acid which was then reutilized. Lactic acid gradually accumulated again at maturity. Enzyme studies on cell-free extracts indicated the presence of all tricarboxylic cycle enzymes except alpha-ketoglutarate dehydrogenase at all stages of development of both RS and OC plants. Included among the enzymes detected were an adenosine monophosphate-stimulated, nicotinamide adenine dinucleotide-isocitric dehydrogenase, and citrate-condensing enzyme. When measured on a per plant basis, tricarboxylic cycle enzyme levels increased during the exponential growth of both kinds of plants. Only after the bicarbonate ceased to have effect on RS plant morphogenesis was there a decrease in the levels of the tricarboxylic cycle enzymes when measured on a per plant basis. Specific activity measurements indicated some differences in the differential rates of synthesis among the enzymes studied previous to 36 hr. Preliminary studies utilizing short periods of (14)C-bicarbonate fixation in young RS plants indicated that during the first 4 min most of the label was located in aspartic acid. These results are discussed in terms of previous results and particularly Cantino's hypothesis concerning the relationship between bicarbonate induction and tricarboxylic-cycle enzymes in the morphogenesis of B. emersonii.
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PMID:Tricarboxylic acid cycle enzymes and morphogenesis in Blastocladiella emersonii. 580 5

Detailed evidence for the amino acid sequence of allosteric citrate synthase from Escherichia coli is presented. The evidence confirms all but 11 of the residues inferred from the sequence of the gene as reported previously [Ner, S. S., Bhayana, V., Bell, A. W., Giles, I. G., Duckworth, H. W., & Bloxham, D. P. (1983) Biochemistry 22, 5243]; no information has been obtained about 10 of these (residues 101-108 and 217-218), and we find aspartic acid rather than asparagine at position 10. Substantial regions of sequence homology are noted between the E. coli enzyme and citrate synthase from pig heart, especially near residues thought to be involved in the active site. Deletions or insertions must be assumed in a number of places in order to maximize homology. Either of two lysines, at positions 355 and 356, could be formally homologous to the trimethyllysine of pig heart enzyme, but neither of these is methylated. It appears that E. coli and pig heart citrate synthases are formed of basically similar subunits but that considerable differences exist, which must explain why the E. coli enzyme is hexameric and allosterically inhibited by NADH, while the pig heart enzyme is dimeric and insensitive to that nucleotide.
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PMID:Amino acid sequence of Escherichia coli citrate synthase. 638 May 76

An alpha-fluoro acid analog and an alpha-fluoro amide analog of acetyl-CoA have been synthesized. The ternary complexes of these inhibitors with oxaloacetate and citrate synthase have been crystallized and their structures analyzed at 1.7 A resolution. The structures are similar to those reported for the corresponding non-fluorinated analogs (Usher et al., 1994), with all forming unusually short hydrogen bonds to Asp 375. The alpha-fluoro amide analog binds with an affinity 1.5-fold lower than that of a previously described amide analog lacking the alpha-fluoro group. The alpha-fluoro acid analog binds with a 50-fold decreased affinity relative to the corresponding unfluorinated analog. The binding affinities are consistent with increased strengths of hydrogen bonds to Asp 375 with closer matching of pKa values between hydrogen bond donors and acceptors. The results do not support any direct correlation between hydrogen bond strength and hydrogen bond length in enzyme-inhibitor complexes.
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PMID:alpha-Fluoro acid and alpha-fluoro amide analogs of acetyl-CoA as inhibitors of citrate synthase: effect of pKa matching on binding affinity and hydrogen bond length. 749 47

The flux through different segments of the tricarboxylic acid cycle was measured in rat brain synaptosomes with gas chromatography-mass spectrometry using either deuterated glutamine or [13C]aspartate. The flux between 2-oxoglutarate and oxaloacetate was estimated to be 3.14 and 4.97 nmol/min/mg protein with and without glucose, respectively. These values were 3-5-fold faster than the flux between oxaloacetate and 2-oxoglutarate (0.92 nmol/min per mg protein) measured in the presence of glucose. The pattern of intermediates labeling suggests that the overall rate-controlling reaction involves either citrate synthase or pyruvate dehydrogenase but not 2-oxoglutarate or isocitrate dehydrogenase. The enrichment in [3,3,4,4-2H4]glutamate from [2,3,3,4,4-2H5]glutamine was as rapid as in [2,3,3,4,4-2H5]glutamate, which indicates that the aspartate aminotransferase reaction is severalfold faster than the flux through the tricarboxylic acid cycle. [13C]Aspartate was rapidly converted to [13C]malate, suggesting that in intact synaptosomes aspartate entry into the mitochondrion is very slow. The finding that aspartate is taken up by mitochondria as malate, along with the observed high enrichment in [3-2H]malate (from [2,3,3,4,4-2H5]glutamine), is consistent with the substantial synaptosomal activity of the malate/aspartate shuttle.
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PMID:Tricarboxylic acid cycle in rat brain synaptosomes. Fluxes and interactions with aspartate aminotransferase and malate/aspartate shuttle. 796 53

The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate. Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively. The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA. However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions. This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent. The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold. Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E. coli enzyme. The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure. The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding. The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.
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PMID:The effect of replacing the conserved active-site residues His-264, Asp-312 and Arg-314 on the binding and catalytic properties of Escherichia coli citrate synthase. 801 Sep 58


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