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)

Limited proteolysis of citrate synthase by Astacus protease, chymotrypsin, clostripain, subtilisin and trypsin on primary fragmentation all yielded similarly sized large (Mr 35 000-36 000) and small fragments (Mr 13 500-14 000) but endoproteinase Lys-C gave fragments of Mr 40 500 and Mr 6500. The sites of the proteolytic attack were determined by Edman degradation of the fragmented synthase preparations, Chymotrypsin, subtilisin, trypsin and endoproteinase Lys-C hydrolyse the synthase at positions 323-324 (-Leu-Arg-), 321-322 (-Ala-Val-)/322-323 (-Val-Leu-), 313-314 (-Arg-Val-) and 366-367 (-Lys-Ala-), respectively. Chymotrypsin and subtilisin attack the small domain of the synthase at the loop between helices O and P very near to a catalytic residue, His-320, and abolish all synthase activities. Primary fragmentation by endoproteinase Lys-C and trypsin reduces the catalytic activity in the physiological overall reaction. Both fragmented enzyme species catalyse the hydrolysis and C-C bond cleavage reactions of citryl-CoA in a stimulated fashion compared to the steady-state rates of the native enzyme, and without hysteretic behaviour. The proteolytic cleavage occurs at acetyl-CoA binding sites within the small domain at the loops connecting helices O to P (trypsin) and Q to R (endoproteinase Lys-C) and reduces the affinity of acetyl-CoA. All of the altered kinetic properties of the fragmented enzyme species are related to this reduced affinity. The correlation between structure and function indicated above is strengthened by the unaltered affinity of oxaloacetate towards the fragmented synthase species. None of the proteolytic enzymes applied attacks oxaloacetate binding sites as defined by the structural work. Oxaloacetate inhibits the hydrolysis of citryl-CoA by the fragmented synthases (endoproteinase Lys-C, trypsin) competitively. An explanation is proposed. The isolated small and large fragments (endoproteinase Lys-C, trypsin) were enzymically inactive. Enzymic activity was restored on recombination of the fragments under denaturing conditions. Cleavage of the loops between helices O to P and Q to R by sequential fragmentation with endoproteinase Lys-C and trypsin inactivated the synthase completely. This result lends support to the idea that the open and closed crystal forms of the structural work are interconverted during the catalytic cycle.
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PMID:Hysteretic behaviour of citrate synthase. Site-directed limited proteolysis. 638 Oct 53

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 was purified to homogeneity from a Gram-positive bacterium (Bacillus megaterium) for the first time. The Mr of the native enzyme was determined to be 84 000 (S.E.M. +/- 5000). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in guanidinium chloride revealed a single protein species of Mr 40 300 (S.E.M. +/- 4400), indicating a dimeric enzyme. This dimeric structure was confirmed by cross-linking the native enzyme with dimethyl suberimidate and with glutaraldehyde, followed by electrophoretic analysis. The enzyme follows Michaelis-Menten kinetics with respect to both substrates, acetyl-CoA and oxaloacetate, and is sensitive to non-specific inhibition by a range of adenine nucleotides. In both molecular and catalytic properties the citrate synthase closely resembles the enzyme from eukaryotic sources and contrasts markedly with the larger, hexameric, enzyme from Gram-negative bacteria.
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PMID:Citrate synthase from a Gram-positive bacterium. Purification and characterization of the Bacillus megaterium enzyme. 641 81

The concentration of metabolically active (i.e. 'free') oxaloacetate in the mitochondrial compartment of isolated liver cells was investigated by two independent approaches. On the basis of mitochondrial aspartate aminotransferase maintaining equilibrium and the direct measurements of mitochondrial aspartate, 2-oxoglutarate and glutamate, the concentration of free oxaloacetate was calculated to be 5 microM after incubation of hepatocytes in the presence of 1.5 mM-lactate and 0.05 mM-oleate. Gradually increasing oleate up to 0.5 mM decreased the free oxaloacetate to 2 microM. Very similar results were obtained when free oxaloacetate concentration was derived from the CO2 production of hepatocytes as a measure of citrate flux through the tricarboxylic acid cycle, and the kinetic data on citrate synthase in situ. The decrease in free oxaloacetate on increasing oleate concentration was associated with lowered rates of cycle-dependent CO2 output and O2 uptake, indicating a decrease in the disposal of acetyl-CoA into the tricarboxylic acid cycle. This decrease could explain 25-30% of the increase in ketone-body production occurring at elevated fatty acid supply. This work documents on a quantitative basis the role of free oxaloacetate in the regulation of ketogenesis.
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PMID:Concentration of free oxaloacetate in the mitochondrial compartment of isolated liver cells. 642 54

Active-site peptides of acetyl transferase, condensing enzyme and acyl carrier protein in the neighborhood of the prosthetic group, 4'-phosphopantetheine, of Cephalosporium caerulens fatty acid synthetase were investigated. The enzyme was reacted with [14C]acetyl-CoA or [14C]iodoacetamide. 14C-Labeled enzyme was digested with pepsin, trypsin or both. 14C-Labeled peptides were isolated by several purification procedures. The amino acid sequence of the active site of condensing enzyme was determined to be Tyr-Gln-Val-Glu-Ser-Cys-Pro-Ile-Leu-Glu-Gly-Lys and that of acetyl transferase was Phe-Ser-Gly-Ala-Thr-Gly-His-Ser-Gln-Gly. The amino acid composition around the 4'-phosphopantetheine-carrying serine was determined to be Asx2, Thr, Ser, Glx3, Gly2, Ala, Ile, Leu3, and Lys. When these active-site peptides were compared with those of Saccharomyces cerevisiae synthetase, a high degree of homology was observed in the active-site peptides of the acetyl transferase and acyl carrier protein domains. However, that of the condensing enzyme domain gave lower homology. These findings may support the assumption that the low reactivity of cerulenin with C. caerulens synthetase is a consequence of the structure of the condensing enzyme domain.
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PMID:Cerulenin resistance in a cerulenin-producing fungus. III. Studies on active-site peptides of fatty acid synthetase from Cephalosporium caerulens. 654 Jul 72

The mechanism of the enzymic reaction responsible for chloramphenicol resistance in bacteria was examined by steady-state kinetic methods. The forward reaction catalysed by chloramphenicol acetyltransferase leads to inactivation of the antibiotic. Use of alternative acyl donors and acceptors, as well as the natural substrates, has yielded data that favour the view that the reaction proceeds to the formation of a ternary complex by a rapid-equilibrium mechanism wherein the addition of substrates may be random but a preference for acetyl-CoA as the leading substrate can be detected. Chloramphenicol and acetyl-CoA bind independently, but the correlation between directly determined and kinetically derived dissociation constants is imperfect because of an unreliable slope term in the rate equation. The reverse reaction, yielding acetyl-CoA and chloramphenicol, was studied in a coupled assay involving citrate synthase and malate dehydrogenase, and is best described by a rapid-equilibrium mechanism with random addition of substrates. The directly determined dissociation constant for CoA is in agreement with that derived from kinetic measurements under the assumption of an independent-sites model.
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PMID:Analysis of the mechanism of chloramphenicol acetyltransferase by steady-state kinetics. Evidence for a ternary-complex mechanism. 659 36

Some mechanism studies on chicken and pig citrate synthase are described. Gibacron Blue F3GA apparently binds into both the oxaloacetate and the acetyl-CoA subsites of the enzyme. Protection by ligands against urea-induced denaturation indicates that several di(tri)-carboxylic acids bind into the oxaloacetate subsite, whereas ATP, but not Mg2+ ATP, binds into the acetyl-CoA subsite. Oxaloacetate, citrate and D-malate induce a transconformation in the enzyme, whereas alpha-ketoglutarate, L-malate and succinate do not.
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PMID:Chicken heart citrate synthase: some mechanism studies. 661 56

S-Dimethylarsino-CoA was synthesized by acylation of CoA with dimethylchloroarsine. The new analogue of acetyl-CoA was tested as an active-site-directed irreversible inhibitor of phosphotransacetylase (EC 2.3.1.8), carnitine acetyltransferase (EC 2.3.1.7) and citrate synthase (EC 4.1.3.7). Irreversible inhibition was observed only with phosphotransacetylase, which was derivatized via a simple bimolecular process (k2 = 197 +/- 15 min-1 . M-1). Acetyl-CoA provided complete substrate protection against the inactivation, while phosphate (a substrate) and desulfo-CoA (a competitive inhibitor) provided a partial protection. The inactivation was not reversed by dithiothreitol. The new reagent was a linear competitive inhibitor versus acetyl-CoA with both carnitine acetyltransferase (Ki = 41 microM) and citrate synthase (Ki = 20 microM). Chemical studies showed that S-dimethylarsino-CoA reacts with the thiol of N alpha-acetylcysteine but not with the side-chain functional groups of histidine and lysine. The nature of the chemical modification of cysteine was determined by investigating a model system. Thus the chemical reaction between the thioarsenite linkage of S-dimethylarsinobenzylmercaptan and the thiol of cysteine was shown to involve transesterification of the dimethylarsino group.
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PMID:Irreversible inhibition of phosphotransacetylase by S-dimethylarsino-CoA. 663 58

A two-step method of determining reduced coenzyme A (CoASH) concentrations in tissue or cell extracts is described. In the first step, CoASH is reacted with acetylphosphate in a reaction catalyzed by phosphotransacetylase to yield acetyl-CoA. Acetyl-CoA is then condensed with [14C]oxaloacetate by citrate synthase to give [14C]citrate. This method allows the measurement of 10-200 pmol of CoASH. By omitting the phosphotransacetylase step, measurement of the same amount of acetyl-CoA is possible.
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PMID:Radioisotopic assay of picomolar amounts of coenzyme A. 665 Aug 21

In this paper, physicochemical evidence is given for the association between the pyruvate dehydrogenase complex (EC 1.2.4.1) and citrate synthase (EC 4.1.3.7) with two gel chromatographic techniques with poly(ethylene glycol) co-precipitation and with ultracentrifugation. Experiments with active enzyme gel chromatography indicate that citrate synthase also associates with pyruvate dehydrogenase complex in its functioning state. Citrate synthase binds to the isolated transacetylase core of pyruvate dehydrogenase complex, but in the binding to the whole pyruvate dehydrogenase complex the two other components of the complex are also involved. One pyruvate dehydrogenase complex can bind 10-11 citrate synthase dimers, and the dissociation constant is about 5.7-6.0 microM as determined by two independent methods. The association between the pyruvate dehydrogenase complex and citrate synthase raises the possibility of the dynamic compartmentation of acetyl-CoA in the mitochondria which results in the direction of acetyl-CoA from pyruvate towards citrate.
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PMID:A study on the physical interaction between the pyruvate dehydrogenase complex and citrate synthase. 665 96


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