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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 of Escherichia coli reacts rapidly with 1 equivalent of Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH. When the enzyme is treated instead with 4,4'-dithiodipyridine (4,4'-PDS), all activity is lost. Certain evidence in this paper is consistent with the belief that the sulfhydryl group modified by DTNB, and that whose modification by 4,4'-PDS inactivates the enzyme, are the same. (i) Both reagents abolish NADH fluorescence enhancement by the enzyme. (ii) Saturating levels of NADH and some other adenylic acid derivatives inhibit the reactions with both reagents. (iii) When the enzyme is modified with one equivalent of DTNB or 4,4'-PDS, subsequent reactivity toward the other reagent is greatly decreased. (iv) Following modifications, the DTNB and 4,4'-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a second enzyme sulfhydryl group, so that an enzyme disulfide is introduced. The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions. Certain evidence has also been found, however, that the sites of modification by DTNB and 4,4'-PDS are not the same. (i) DTNB modification desensitizes to NADH but does not inactivate, while 4,4'-PDS inactivates at least 99.9%. (ii) The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4'-PDS modified product. (iii) Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4'-PDS. The studies therefore indicate a close relationship between the DTNB desensitization and 4,4'-PDS inactivation, but they are unable to identify it exactly. Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB.
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PMID:The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and 4,4'-dithiodipyridine. 3 91

The conformational stabilities of native pig citrate synthase (PCS), a recombinant wild-type PCS, and six active-site mutant pig citrate synthases were studied in thermal denaturation experiments by circular dichroism and in urea denaturation experiments by using DTNB to measure the appearance of latent SH groups. His274 and Asp375 are conserved active-site residues in pig citrate synthase that bind to substrates and are implicated in the catalytic mechanism of the enzyme. By site-directed mutagenesis, His274 was replaced with Gly and Arg, while Asp375 was replaced with Gly, Asn, Glu, or Gln. These modifications were previously shown to result in 10(3)-10(4)-fold reductions in enzyme specific activities. The thermal unfolding of pig citrate synthase and the six mutants in the presence and absence of substrates showed large differences in the thermal stabilities of mutant proteins compared to the wild-type pig citrate synthase. The functions of His274 and Asp375 in ligand binding were measured by oxalacetate protection against urea denaturation. These data indicate that active-site mutations that decrease the specific activity of pig citrate synthase also cause an increase in the conformational stability of the protein. These results suggest that specific electrostatic interactions in the active site of citrate synthase are important in the catalytic mechanism in the chemical transformations as well as the conformational flexibility of the protein, both of which are important for the overall catalytic efficiency of the enzyme.
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PMID:Conformational stability of pig citrate synthase and some active-site mutants. 189 35

Citrate synthase (EC 4.1.3.7) from Tetrahymena pyriformis has been purified 185-fold. The molecular weight of the native enzyme was determined to be 120,000. The enzyme is labile at low ionic strength, but can be stabilized by KCl and glycerol. It is activated by KCl at low (below 60 mM) or high concentrations, and inhibited by divalent cations (Mn2+, Mg2+, Ca2+). The Michaelis constants are 0.1 mM for oxalacetate and 0.01 mM for acetyl-CoA. The kinetics with oxalacetate exhibit negative cooperativity, with a nH = 0.66. Among the metabolites tested, only ATP and GTP can inhibit the enzyme but Mg2+ relieves the ATP inhibition. Incubation with sulfhydryl reagents (DTNB) in the absence of its substrates results in a rapid inactivation of the enzyme. It is concluded that Tetrahymena citrate synthase is closer to the enzyme from Gram-positive bacteria than to those of eucaryotes.
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PMID:Citrate synthase of Tetrahymena pyriformis: evolutionary and regulatory aspects. 640 83

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

Muscle carnitine levels were examined in 31 younger [mean (SD), 27 (5) years] and 27 older [49 (8) years] men. Needle biopsies were obtained from the lateral gastrocnemius or vastus lateralis muscles and assayed for free and total carnitine concentrations via a 5,5'-Dithiobis-(2-nitrobenzoic acid) DTNB-linked spectrophotometric procedure. A subgroup of subjects (n = 28) were assessed for citrate synthase (CS) and succinate dehydrogenase (SDH) activity, and type I muscle fiber composition (% type I fibers). An additional sub-group of nine subjects was assessed for free and total serum carnitine levels. No mean (SEM) differences in free [21.6 (0.7) vs 20.3 (0.9) mumol.g dry weight-1] and total [26.4 (0.6) vs 26.1 (0.9) mumol.g dry weight-1) muscle carnitine levels were found between the younger and older subjects, respectively. Correlational data revealed no significant relationships between total muscle carnitine and CS (r = -0.36), SDH (r = -0.26), or % type I fibers (r = -0.16). In addition, there was a low non-significant relationship between serum and muscle total carnitine concentrations (r = -0.44). These findings suggest that muscle carnitine levels are similar between younger and older males, and there does not appear to be any relationship between muscle carnitine and markers of muscle oxidative potential (i.e., oxidative enzymes, % type I fiber). Since serum carnitine is often used as an indicator of body carnitine status, it is noteworthy that we found a low negative relationship between blood and muscle carnitine concentrations.
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PMID:Relationships between muscle carnitine, age and oxidative status. 758 81

In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutant Thermoplasma citrate synthase is very similar to wild-type citrate synthase in its substrate and co-factor specificities, pH profile and thermal stability. The mutation, however, has decreased the enzyme activity. The newly introduced reactive sulphydryl group could be easily modified by DTNB and labelled with 4-chloro-7-sulphobenzofuran, without loss of any activity.
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PMID:The effect of cysteine-43 mutation on thermostability and kinetic properties of citrate synthase from Thermoplasma acidophilum. 869 16