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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 from Acinetobacter calcoaceticus was subjected to proteolysis with subtilisin. Although the enzyme proved relatively resistant to inactivation by this treatment, SDS-polyacrylamide gel electrophoresis clearly revealed breakdown of the citrate synthase to smaller fragments. The regulatory responses of the native enzyme to inhibition by NADH and re-activation by AMP were retained on proteolysis, indicating that the fragments bind tightly to each other and preserve the overall cooperative molecular interactions.
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PMID:Proteolysis of acinetobacter citrate synthase by subtilisin. 633 74

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

NADH:ubiquinone reductase (complex I) of the mitochondrial inner membrane respiratory chain binds a number of mitochondrial matrix NAD-linked dehydrogenases. These include pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, mitochondrial malate dehydrogenase, and beta-hydroxyacyl-CoA dehydrogenase. No binding was detected between complex I and cytosolic malate dehydrogenase, glutamate dehydrogenase, NAD-isocitrate dehydrogenase, lipoamide dehydrogenase, citrate synthase, or fumarase. The dehydrogenases that bound to complex I did not bind to a preparation of complex II and III, nor did they bind to liposomes. The binding of pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and mitochondrial malate dehydrogenase to complex I is a saturable process. Based upon the amount of binding observed in these in vitro studies, there is enough inner membrane present in the mitochondria to bind the dehydrogenases in the matrix space. The possible metabolic significance of these interactions is discussed.
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PMID:Complex I binds several mitochondrial NAD-coupled dehydrogenases. 643 16

A mathematical model is proposed to describe the interaction between glycolysis, the Krebs cycle and 3-oxidation (beta OX). The model incorporates the activations of phosphofructokinase by AMP and of isocitrate dehydrogenase by ADP as well as the inhibitions of citrate synthase by citrate, of acyl CoA synthase by excess CoAsAcyl, of pyruvate dehydrogenase (PDH) and the beta OX helix by the products CoAsAc and NADH. These regulations have been shown to provide consecutive triggering of the fatty acid and glucose oxidation systems with an increase in the ATPase load, the beta OX of fatty acids being a major source of energy at small loads. The steady state rates of glycolysis and PDH-reaction begin to increase at larger loads when the rate of beta OX is close to its maximum value. At maximum ATPase loads, the glucose oxidation accounts for more than 80% of the total energy production. Under limited fatty acid supply, the transfer to glucose oxidation gives rise to a region of the ATPase loads, where in the steady state levels of NADH and CoAsAc increase with load.
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PMID:[Ratio between carbohydrate and lipid metabolism in muscle cell energy metabolism during ATPase loading. Mathematical model]. 645 74

By a cellular subfractionation technique, synaptic and non-synaptic mitochondria from a single rat cerebral cortex were obtained. In these different mitochondrial populations the activity of citrate synthase, malate dehydrogenase, total NADH-cytochrome c reductase, cytochrome oxidase and glutamate dehydrogenase were evaluated. Except for glutamate dehydrogenase, the enzyme specific activities evaluated in the "free" mitochondrial fraction were higher than those evaluated in the "synaptic" SM1 and SM2 mitochondrial fractions, the differences between SM1 and SM2 fractions being significant. The effect of the in vivo administration of naftidrofuryl given at different doses and at different times was studied. The treatment induced few but different changes in the various mitochondrial populations.
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PMID:Synaptic and non-synaptic mitochondria from rat cerebral cortex. Characterization and effect of pharmacological treatment on some enzyme activities related to energy transduction. 661 53

The binding of two similar spin-labeled fatty acyl-CoA analogues, one short chain, 6-doxyloctanoyl-CoA (S-(2-(5-carboxybutyl)-2-ethyl-4, 4-dimethyl-3-oxazolidinyl-N-oxyl)-CoA) and one long chain, 6-doxylstearoyl-CoA (S-(2-(5-carboxybutyl)-2-dodecyl-4, 4-dimethyl-3-oxazolidinyl-N-oxyl)-CoA) to pig heart citrate synthase (citrate oxaloacetate-lyase (pro-3S-CH2COO- leads to acetyl-CoA) EC 4.1.3.7) has been compared. The binding of the short chain analogue could be satisfactorily fit by a classical treatment (independent, noninteracting sites) with well defined stoichiometry: 2 mol of spin label bound per mol of dimeric enzyme. Binding of the long chain analogue was complex and in excess of 2 mol/dimer. Competitive binding experiments using either analogue in the presence of various nucleotides and substrates revealed differences in the binding of the long and short chain analogues. These additional studies, together with kinetic measurements, implied isosteric binding of acyl-CoA, ATP, NADPH, NADH, NADP+, acetyl-CoA, and partial isosteric binding of the long chain acyl-CoA. Binding of NADPH and NADP+ to the same form of the enzyme, perhaps through overlapping sites, was kinetically verified even though these nucleotides had differing effects on the binding of the spin-labeled analogues. Oxalacetate was shown to decrease the binding of the long chain analogue but to have no effect on the binding of the short chain. This result was supported by kinetic measurements. The competitive binding experiments with the long chain analogue suggested that its complex isotherm resulted from binding in two classes of sites, i.e. two cooperative nucleotide sites and other sites. An empirical mathematical model employing this rationale provided a satisfactory fit for the binding of fatty acyl-CoA to citrate synthase. A spin-labeled fatty acid which was not bound by the native enzyme was appreciably bound in the presence of additional palmitoyl-CoA. This binding might be identified with one of the two sets of binding sites proposed in the model. These and previous results on acyl-CoA binding were correlated with the properties of the CoA binding site defined crystallographically (Remington, S., Wiegand, G., and Huber, R. (1982) J. Mol. Biol. 158, 111-152).
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PMID:Regulation of enzymes by fatty acyl coenzyme A. Interactions of short and long chain spin-labeled acyl-CoA with the acetyl-CoA site on pig heart citrate synthase. 669 13

Pig heart citrate synthase was subjected to limited proteolytic attack by subtilisin, chymotrypsin, and trypsin in the presence of palmitoyl-CoA. Initial proteolysis by all three proteolytic enzymes resulted in cleavage of the monomeric subunit (Mr 45 000 +/- 3000) into a large (Mr 35 000-38 500) and a small (Mr 9000 +/- 3000) into a large (Mr 35 000-38 500) and a small (Mr 9000-12 000) fragment. Further proteolysis of the large subunit produced a secondary fragment (Mr 31 000-36 000). The small (Mr 9000-12 000) fragment was stable in the presence of subtilisin but was substantially degraded by both chymotrypsin and trypsin. The actual molecular weight of fragments varied with the choice of the proteolytic enzyme. Limited proteolysis was absolutely dependent on the presence of palmitoyl-CoA and resulted in complete inhibition of the catalytic activity of the enzyme. Citrate, ammonium sulfate, and especially oxaloacetate provided complete protection against proteolysis whereas acetyl-CoA, CoASH, NADH, and ATP were ineffective. Reaction of rabbit anti-citrate synthase with citrate synthase and its proteolytic fragments indicated that the main antigenic region lay primarily in the small fragment. The products of subtilisin cleavage were isolated by gel filtration under denaturing conditions. The large (Mr 35 000-38 500) fragment contained the amino-terminal (approximately)336 amino acids and the small fragment contained the remaining carboxyl-terminal amino acids. The results are discussed in relation to the structure of citrate synthase.
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PMID:Limited proteolysis of pig heart citrate synthase by subtilisin, chymotrypsin, and trypsin. 677 58

Citrate synthase has been purified to homogeneity from a strain of the Gram-negative aerobic bacterium Acinetobacter anitratum in a form which retains its sensitivity to the allosteric inhibitor NADH. In subunit size, amino acid composition, and antigenic reactivity the enzyme shows a marked structural resemblance to the citrate synthase of the Gram-negative facultative anaerobe Escherichia coli. Whereas the E. coli enzyme is subject to a strong, hyperbolic inhibition by NADH (Hill's number n = 1.0, Ki = 2 microM), the A. anitratum enzyme shows a weak, sigmoid response (n = 1.6, I0.5 = 140 microM) to this nucleotide. With E. coli, NADH inhibition is competitive with acetyl-CoA, and noncompetitive with oxaloacetate; with A. anitratum, NADH is noncompetitive with both substrates. Acinetobacter anitratum citrate synthase shows hyperbolic saturation with acetyl-CoA (n = 1.8). The finding of Weitzman and Jones (Nature (London) 219, 270 (1968) that NADH inhibition of the enzyme from Acinetobacter spp. is reversible by AMP, while that from E. coli is not, is explained by the much greater affinity of the E. coli enzyme for NADH. Unlike E. coli citrate synthase, the A. anitratum enzyme does not react with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of denaturation. With a second sulfhydryl reagent, 4,4'-dithiodipyridine (4,4'-PDS), the A. anitratum enzyme reacts with 1 equiv. of subunit; this modification induces a partial activity loss (attributable to a arise in the Km for acetyl-CoA) and an increase in the sensitivity to NADH. With the E. coli enzyme, 4,4'-PDS causes complete inactivation. Acinetobacter anitratum citrate synthase is much more resistant to urea denaturation than the E. coli enzyme is; the resistance of both enzymes to urea is greatly improved in the presence of 1 M KCl. It is suggested that the amino acid sequences of the subunits of the citrate synthases of these two bacteria are about 90% homologous, and that the 10% differences are in key residues, perhaps largely in the subunit contact regions, which account for the differences in allosteric properties.
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PMID:A comparison of the citrate synthases of Escherichia coli and Acinetobacter anitratum. 678 Jan 70

Citrate synthase [citrate (si)-synthase] (EC 4.1.3.7) was partially purified from extracts of highly purified typhus rickettsiae (Rickettsia prowazekii). Molecular exclusion and affinity column chromatography were used to prepare 200-fold-purified citrate synthase that contained no detectable malate dehydrogenase (EC 1.1.1.37) activity. Rickettsial malate dehydrogenase also was partially purified (200-fold) via this purification procedure. Catalytically active citrate synthase exhibited a relative molecular weight of approximately 62,000 after elution from a calibrated Sephacryl S-200 column. Acetyl coenzyme A saturation of partially purified enzyme was sensitive to strong competitive inhibition with adenylates (ATP greater than ADP much greater than AMP). [beta,gamma-methylene]ATP, dATP, and dADP also caused strong inhibition, but guanosine and cytosine nucleotides were significantly less inhibitory. Adenylates had no effect on oxalacetate saturation kinetics when acetyl coenzyme A was present in high concentration (greater than or equal to 50 microM). Neither NADH nor alpha-ketoglutarate affected the saturation kinetics of rickettsial citrate synthase. Thus, citrate synthase from R. prowazekii exhibits greater similarity to the eucaryotic and gram-positive procaryotic enzymes than to citrate synthase from free-living gram-negative bacteria. These results represent the first characterization of a highly purified key regulatory enzyme from these obligate intracellular parasitic bacteria.
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PMID:Regulatory properties of citrate synthase from Rickettsia prowazekii. 679 96

Experiments performed in polyethylene glycol and with a divalent crosslinker indicate that both mitochondrial malate dehydrogenase and aspartate aminotransferase can form hetero enzyme--enzyme complexes with either glutamate dehydrogenase or citrate synthase. In general, these as previous results indicate that complexes with the aminotransferase are favored over those with malate dehydrogenase and complexes with glutamate dehydrogenase are favored over those with citrate synthase. When the levels of enzymes are low, the only detectable complex is between the aminotransferase and glutamate dehydrogenase. Under these conditions, palmitoyl-CoA is required for complexes between the other three enzyme pairs, however, palmitoyl-CoA also enhances interactions between glutamate dehydrogenase and the aminotransferase. DPNH disrupts complexes with malate dehydrogenase and has little effect on those with the aminotransferase, while oxalacetate disrupts complexes with citrate synthase but has little effect on those with glutamate dehydrogenase. The citrate synthase-aminotransferase complex was favored in the presence of DPNH plus malate, which disrupt the other three enzyme-enzyme complexes. Glutamate dehydrogenase has a higher affinity and capacity than citrate synthase for palmitoyl-CoA. Consequently, lower levels of palmitoyl-CoA are required to enhance interactions with glutamate dehydrogenase. Furthermore, glutamate dehydrogenase can compete with citrate synthase for palmitoyl-CoA and thus can prevent palmitoyl-CoA from enhancing interactions between citrate synthase and either malate dehydrogenase or the aminotransferase.
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PMID:Complexes between mitochondrial enzymes and either citrate synthase or glutamate dehydrogenase. 682 31


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