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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)

Deviations from Michealis-Menten kinetics in the pig-heart citrate synthase (citrate-oxaloacetate-lyase(pro-3S-CH2-COO-leads to acetyl-CoA), EC 4.1.3.7) system have been characterized and analyzed in view of the kinetic theory described in the preceding paper. The enzymic condensation reaction between acetyl-CoA and oxaloacetate is subject to substrate-inhibition by acetyl-CoA. This can be attributed to the formation of a productive enzyme-acetyl-CoA complex with a dissociation constant of 110 uM. The binding of acetyl-CoA to the enzyme decreases the on-velocity constant for oxaloacetate-binding from 4000 min-1- micrometer-1 to 1700 min-1-micrometer-1. The affinity of citrate synthase for oxaloacetate increase at least 20-fold on the binding of acetyl-CoA. The latter cooperativity effect can be attributed to a more than 45-fold decrease of the off-velocity constant for oxaloacetate-binding.
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PMID:Substrate-inhibiton by acetyl-CoA in the condensation reaction between oxaloacetate and acetyl-CoA catalyzed by citrate synthase from pig heart. 56 Aug 67

In isolated hepatocytes from normal fed rats, the subcellular distribution of malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH has been determined by a modified digitonin method. Incubation with various substrates (lactate, pyruvate, alanine, oleate, oleate plus lactate, ethanol and aspartate) markedly changed the total cellular amounts of metabolites, but their distribution between the cytosolic and mitochondrial compartments was kept fairly constant. In the presence of lactate, pyruvate or alanine, about 90% of cellular aspartate, malate and oxaloacetate, and 50% of citrate was located in the cytosol. The changes in acetyl-CoA in the cytosol were opposite to those in the mitochondrial space, the sum of both remaining nearly constant. The mitochondrial acetyl-CoA/CoASH ratio ranged from 0.3-0.9 and was positively correlated with the rate of ketone body formation. The mitochondrial/cytosolic (m/c) concentration gradients for malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH averaged from hepatocytes under different substrate conditions were determined to be 1.0, 8.8, 1.6, 2.2, 0.5, 0.7, 13 and 40, respectively. From the distribution of citrate, a pH difference of 0.3 across the inner mitochondrial membrane was calculated, yet lower values resulted from the m/c gradients of 2-oxoglutarate, glutamate and malate. The mass action ratios for citrate synthase and mitochondrial aspartate aminotransferase have been calculated from the metabolite concentrations measured in the mitochondrial pellet fraction. A comparison with the respective equilibrium constants indicates that in intact hepatocytes, neither enzyme maintains its reactants at equilibrium. On the assumption that mitochondrial malate dehydrogenase and 3-hydroxybutyrate dehydrogenase operate near equilibrium, the concentration of free oxaloacetate appears to be 0.3-2 micron, depending on the substrate used. Plotting the calculated free mitochondrial oxaloacetate concentration against the citrate concentration measured in the mitochondrial pellet yielded a hyperbolic saturation curve, from which an apparent Km of citrate synthase for oxaloacetate in the intact cells of 2 micron can be derived, which is comparable to the value determined with purified rat liver citrate synthase. The results are discussed with respect to the supply of substrates and effectors of anion carriers and of key enzymes of the tricarboxylic acid cycle and fatty acid biosynthesis.
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PMID:Distribution of metabolites between the cytosolic and mitochondrial compartments of hepatocytes isolated from fed rats. 68 Jun 39

Hepatic citrate synthase activity has been shown to be increased 2- to 3-fold in vitamin B12 deficiency. Immunochemical titrations of the affinity chromatography-purified enzyme obtained from liver of animals with B12 deprivation demonstrated that this increase in activity was the result of a true increase in enzyme protein content. When fixed ratios of aliquots of normal and B12-deprived rat liver homogenates were mixed, the activity measured showed no change from the expected total citrate synthase activity based on the admixture ratios. Partial purification of the enzyme resulted in the expected recovery of the enzyme at each of the purification steps. Thus, it is unlikely that the change in enzyme activity in B12 deprivation was due to the presence of a soluble or easily dissociable normally occurring activator or inhibitor. Ouchterlony double diffusion studies, immunochemical titration, and determination of Km vlaues for exalacetate and acetyl-CoA (substrates for citrate synthase) and Ki values for ATP (inhibitor of citrate synthase) all indicated that the enzyme from the B12-deprived livers was structurally the same as that from normal liver. Hepatic citrate synthase degradation rate constants were shown to be essentially unchanged in B12deficiency. The rate of hepatic citrate synthase synthesis, under steady state conditions, was shown to be 2.8-fold greater in the B12-deficient animal than in the normal animal. The increased rate of synthesis appeared to explian the increased enzyme content. Finally, no change in specific activity of the enzyme was seen in brain, heart, or kidney in the B12-deprived animal.
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PMID:Studies of the mechanism by which hepatic citrate synthase activity increases in vitamin B12 deprivation. 81 82

A comparative study of the citrate synthases purified from the facultatively photosynthetic bacterium Rhodospirillum rubrum (Gram negative) and the thermophile Bacillus stearothermophilus (Gram positive) was made. The citrate synthase from R. rubrum was activated by KCl (6-fold at 0.1 M KCl) and, less effectively, by NaCl and NH4Cl. Its molecular weight was about 300,000. The enzyme was strongly inhibited by NADH, and this inhibition was counteracted by AMP. The citrate synthase from B. stearothermophilus was little affected by KCl, NaCl and NH4Cl, all of which activated by about 25% at 0.1 M. Its molecular weight was ca 100,000. The enzyme was not affected by NADH or AMP. Both citrate synthases were insensitive to alpah-oxoglutarate concentrations up to 5 mM, and were inhibited by ATP; the B. stearothermophilus enzyme was more strongly inhibited than the R. rubrum enzyme. In both cases the ATP inhibition was strictly competitive towards acetyl-CoA and non-competitive towards oxaloacetate. Both enzymes, in spite of the peculiar physiological properties of their bacterial sources, followed the close correlation between the properties of the citrate synthase and the taxonomical position of the microorganism, proposed by Weitzman and his co-workers.
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PMID:[Regulation of citrate synthese in bacteria: Comparison of the action of various effectors on the enzymes of Rhodospirillum rurbum and Bacillus stearothermophilus]. 82 87

The NH2 terminus of ovalbumin is acetylated in cell-free protein-synthesizing systems as it is in vivo. The acetyl group is derived from acetyl-CoA and it is incorporated during translation. Acetylation can be prevented by metabolizing the available acetyl-CoA to citrate with the addition of citrate synthase and oxalacetate to the translation system. The NH2 terminus of ovalbumin synthesized under these conditions can be sequenced by automated Edman degradation. This procedure has also been applied to the sequencing of Pr 76gag, the viral core protein precursor synthesized from 35 S Rous sarcoma virus RNA.
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PMID:Prevention of NH2-terminal acetylation of proteins synthesized in cell-free systems. 92 22

The activity of "satellite" enzymes related to gluconeogenesis has been measured in the oocytes and embryos at the early stages of loach (Misgurnus fossilis L.) embryogenesis. The activity of pyruvate dehydrogenase increase during oocyte maturation by 30%, remains constant at the cleavage and blastula stages and decreased on the onset of gastrulation. In the both oocytes and embryos pyruvate dehydrogenase has been found only in the active form. The activity of citrate synthase, malate dehydrogenase and pyruvate carboxylase remained constant during oocyte maturation and et all early stage of embrional development. Citrate lyase and "malic"-enzyme were not found, Oocyte maturation is followed by a considerable increase in the malate and oxalacetate content, the level of pyruvate and acetyl-CoA being found invariable.
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PMID:[Characteristics of the activity of "satellite" enzymes of gluconeogenesis in the oocytes and embryos of loach]. 103 Jun 40

The steady state mitochondrial content of coenzyme A-SH (CoA), acetyl-CoA, succinyl-CoA, and long chain acyl-CoA has been determined during the oxidation of palmitoylcarnitine by rabbit heart mitochondria. Variation of the substrate concentration during ADP-stimulated (state 3) respiration varies the mitochondrial content of long chain acyl-CoA and the rate of O2 uptake, and permits the conclusion that the Km of beta oxidation for intramitochondrial long chain acyl-CoA is approximately 1 nmol/mg of mitochondrial protein. At near saturating concentrations of palmitoylcarnitine, plus L-malate, the addition of ADP causes a decrease in acetyl-CoA, an increase in CoA and succinyl-CoA, and no clear change in long chain acyl-CoA content. These changes reverse upon the depletion of ADP (state 3 leads to 4 transition). Similar changes in CoA, acetyl-CoA, and succinyl-CoA are seen during state 4 leads to 3 leads to 4 transitions with pyruvate plus L-malate and octanoate plus L-malate as substrates. These results suggest a limitation of flux by citrate synthase during the controlled oxidation of these three substrates. The ratio acetyl-CoA/succinyl-CoA was determined not only during state 3 and state 4 oxidation of palmitoylcarnitine plus L-malate and pyruvate plus L-malate, but also during intermediate respiratory states (state 3 1/2) generated by adding glucose and varying amounts of hexokinase. These intermediate states are characterized by a high succinyl-CoA content, relative to either state 3 or state 4, and a suboptimal flux through citrate synthase, estimated either by pyruvate disappearance or by O2 uptake.
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PMID:The steady state concentrations of coenzyme A-SH and coenzyme A thioester, citrate, and isocitrate during tricarboxylate cycle oxidations in rabbit heart mitochondria. 119 59

The synthesis of ketone bodies by intact isolated rat-liver mitochondria has been studied at varying rates of acetyl-CoA production and of acetyl-CoA utilization in the Krebs cycle. Factors which enhanced the rate of acetyl-CoA production caused an increase in the fraction of acetyl-CoA which was incorporated into ketone bodies. On the other hand, it was found that factors which stimulated the formation of citrate lowered the relative rate of ketogenesis. It is concluded that acetyl-CoA is preferentially used for citrate synthesis, if the level of oxaloacetate in the mitochondrial matrix space is adequate. The intramitochondrial level of oxaloacetate, which is determined by the malate concentration and the ratio of NADH over NAD+, is the main factor controlling the rate of citrate synthesis. The ATP/ADP ratio per se does not affect the activity of citrate synthase in this in vitro system. Ketogenesis can be described as an overflow of acetyl-groups: Ketone-body formation is stimulated only when the rate of acetyl-CoA production increases beyond the capacity for citrate synthesis. The interaction between fatty acid oxidation and pyruvate metabolism and the effects of long-chain acyl-CoA on mitochondrial metabolism are discussed. Ketone bodies which were generated during the oxidation of [1-14C] fatty acids were preferentially labelled in their carboxyl group. This carboxyl group had the same specific activity as the acetyl-CoA pool, whereas the specific activity of the acetone moiety of acetoacetate was much lower, especially at low rates of ketone-body formation. The activities of acetoacetyl-CoA deacylase and the hydroxymethylglutaryl-CoA (HMG-CoA) pathway were compared in soluble and mitochondrial fractions of rat- and cow-liver in different ketotic states. In rat-liver mitochondria, both pathways of acetoacetate synthesis were stimulated upon starvation or in alloxan diabetes. In cow liver, only the HMG-CoA pathway was increased during ketosis in the mitochondrial as well as in the soluble fraction.
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PMID:Aspects of ketogenesis: control and mechanism of ketone-body formation in isolated rat-liver mitochondria. 119 5

Feedback control between flux through the phosphorylating electron transport chain and the coordination of flux through individual steps of the citric acid cycle have been investigated under a number of different conditions of substrate availability and workloads in the isolated perfused rat heart. The transition from substrate-free perfusion to perfusion with glucose and insulin with no change of workload was associated with increases in the pool sizes of citric acid cycle intermediates except for oxaloacetate, but with an initial imbalance of flux through individual steps in the cycle and transport of anions of the malate-aspartate cycle across the mitochondrial membrane. Flux through citrate synthase initially increased while that through alpha-ketoglutarate dehydrogenase decreased. Of the components of the malate-aspartate cycle, flux through the malate-alpha-ketoglutarate exchange was increased prior to that through the glutamate-aspartate exchange and intramitochondrial aspartate aminotransferase. These changes can be accounted for on the basis of known kinetic controls of the enzyme and transport steps in response to increased pyruvate, acetyl-CoA, and NADH delivery at an approximately constant rate of ATP turnover.
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PMID:Coordination of citric acid cycle activity with electron transport flux. 126 91

Citrate synthase complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CM-CoA), are believed to mimic those with the activated form of acetyl-CoA. The X-ray structure [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213] of the ternary complex of the enzyme, oxaloacetate, and CMCoA has been used as the basis for a proposal that a neutral enol of acetyl-CoA is that activated form. Since the inhibitor carboxyl has a pKa of 3.90, analogy with an enolic acetyl-CoA intermediate leads to the prediction that a proton should be taken up from solution upon formation of the analog complex so that the transition-state analog carboxyl is protonated when bound. We have obtained evidence in solution for this proposal by comparing the isoelectric points and the pH dependence of the dissociation constants of the ternary complexes of the pig heart enzyme with the neutral ground-state analog inhibitor, acetonyl-CoA (KCoA), and the anionic transition-state analog inhibitor (CMCoA) and by studying the NMR spectra of the transition-state analog complexes of allosteric (Escherichia coli) and nonallosteric (pig heart) enzymes. The pH dependence of the dissociation constant of the ground-state analog indicates no proton uptake, while that for the transition-state analog indicates that 0.55 +/- 0.04 proton is taken up when the analog binds to the citrate synthase-oxaloacetate binary complex. The overall charges of ternary complexes of the pig heart enzyme with the transition-state and ground-state analog inhibitors are the same, as monitored by their isoelectric points.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proton uptake accompanies formation of the ternary complex of citrate synthase, oxaloacetate, and the transition-state analog inhibitor, carboxymethyl-CoA. Evidence that a neutral enol is the activated form of acetyl-CoA in the citrate synthase reaction. 132 22


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