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)

Current evidence suggests that mitochondrial matrix enzymes exist in solid-state, multienzyme complexes in vivo. Addition of polyethylene glycol to a solution containing malate dehydrogenase and citrate synthase generates such a solid-state, enzyme complex in vitro at enzyme concentrations permitting kinetic measurements. Suspensions of the isolated, solid-state, hetero-complex of these enzymes were used to study the coupled reactions of citrate synthesis from malate, NAD, and CoASAc. The particles appear to be about 1 microgram in diameter. Considering the ratio of enzyme to oxalacetate molecules in or at the surface of the solid-state particles, one would expect oxalacetate to be converted to citrate within a few molecular distances of the site of oxalacetate generation. This model of "substrate channeling" (or alternatively a direct transfer of oxalacetate between enzymes) is supported by experiments with excess aspartate aminotransferase and glutamate added to the solution phase to give a reaction competing with the synthase for bulk phase oxalacetate. Quantities of aminotransferase that reduce the citrate reaction rate with soluble dehydrogenase and synthase by 90% do not significantly affect rates with comparable amounts of the dehydrogenase-synthase complex. We suggest that similar substrate channeling can occur in vivo and discuss the possible advantages provided thereby.
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PMID:Substrate channeling of oxalacetate in solid-state complexes of malate dehydrogenase and citrate synthase. 406 62

An immobilized three-enzyme system, malate dehydrogenase (EC 1.1.1.37)-citrate synthase (EC 4.1.3.7)-lactate dehydrogenase (EC 1.1.1.27), was investigated as a model for the rate of oxalacetate production and utilization in mitochondria. Lactate dehydrogenase is included to mimic the NADH-utilizing system of mitochondria. This three-enzyme system was immobilized in three different ways (1) on Sephadex G-50 (surface coupling), (2) on Sepharose 4B (internal-external coupling), and (3) entrapped in polycrylamide gel. The rate of citrate production from malate, NAD(+), and acetyl CoA was determined continuously in a flow system. Up to about 100% rate enhancements were observed when the immobilized system was compared to identical systems of free enzyme. An even more pronounced increase of rate of up to about 400% compared to the soluble system was measured after addition of pyruvate (to reoxidize formed NADH). These results are interpreted in relation to microenvironmental changes of oxalacetate production and the possible organization of enzymes of the Krebs cycle.
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PMID:An immobilized three-enzyme system: a model for microenvironmental compartmentation in mitochondria. 435 55

The synthesis of citric and glutamic acids by extracts of Chloropseudomonas ethylicum was studied with labeled precursors. When acetyl-coenzyme A-1-(14)C was used as substrate, only 0.1% of the total radioactivity was found in the C-5 position of citric acid; whereas, with oxalacetate-4-(14)C as substrate, 100% of the total radioactivity was found in C-5. These results demonstrated that the Chloropseudomonas citrate synthetase had an absolute stereospecificity, identical to that of the pig heart synthetase. The distribution of radioactivity in the glutamic acid synthesized from acetyl-coenzyme A-1-(14)C was 0% in C-1 and 94.0% in C-5; whereas the glutamic acid formed from oxalacetate-4-(14)C contained 89.6% in C-1 and 0.5% in C-5. This distribution is entirely consistent with the biosynthesis of glutamic acid from citric acid via aconitase, d(s)-isocitrate, and l-glutamate dehydrogenases. The presence of l-glutamate dehydrogenase in extracts was demonstrated. The stereospecificity of the citrate synthetase and the pattern of glutamate labeling further establish that the aconitase of Chloropseudomonas is completely stereospecific.
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PMID:Stereospecificity of citrate synthetase in relation to glutamate biosynthesis by extracts of Chloropseudomonas ethylicum. 564 42

Burton, Sheril D. (Institute of Marine Science, University of Alaska, College), Richard Y. Morita, and Wayne Miller. Utilization of acetate by Beggiatoa. J. Bacteriol. 91:1192-1200. 1966.-A proposed system which would permit acetate incorporation into four-carbon compounds without the presence of key enzymes of the citric acid cycle or glyoxylate cycle is described. In this system, acetyl-coenzyme A (CoA) is condensed with glyoxylate to form malate, which, in turn, is converted to oxaloacetate. Oxaloacetate then reacts with glutamate to produce alpha-ketoglutarate, which is subsequently converted to isocitrate. Cleavage of isocitrate produces glyoxylate and succinate. Thus, the proposed system is similar to the glyoxylate bypass in that malate is produced from glyoxylate and acetyl-CoA, but differs from both the citric acid cycle and the glyoxylate bypass, since citrate and fumarate are not involved. Fumarase, aconitase, catalase, citritase, pyruvate kinase, enolase, phosphoenolpyruvate carboxylase, lactic dehydrogenase, alpha-ketoglutarate dehydrogenase, and condensing enzyme were not detectable in crude extracts of Beggiatoa. Succinate was oxidized by a soluble enzyme not associated with an electron-transport particle. Isocitrate was identified as the sole compound labeled when C(14)O(2) was added to a reduced nicotinamide adenine dinucleotide, CO(2) generating system (crystalline glucose-6-phosphate dehydrogenase and glucose-6-phosphate) in the presence of alpha-ketoglutarate.
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PMID:Utilization of acetate by Beggiatoa. 592 51

Cell-free extracts of Acetobacter suboxydans were prepared which were capable of condensing alpha-ketoisovalerate with (14)C-labeled acetyl-coenzyme A to yield (14)C-labeled alpha-isopropylmalate. The product of the reaction was isolated by paper and column chromatography and was characterized by recrystallization with synthetic alpha-isopropylmalic acid to constant specific radioactivity. The formation of alpha-isopropylmalate by extracts of A. suboxydans plus the ability of the organism to grow in a simple glucose-glycerol medium containing glutamic acid as the only amino acid indicate that the pathway for leucine biosynthesis shown to exist in yeast and Salmonella typhimurium also occurs in A. suboxydans. As a comparison, the condensation of oxalacetate and ((14)C) acetyl-coenzyme A to yield ((14)C) citric acid was shown, by similar means, to occur in A. suboxydans. This is of interest since the existence of this classical condensing enzyme has hitherto not been demonstrated in this organism. This reaction was further demonstrated in cell-free extracts of A. suboxydans by means of a spectrophotometric assay at 232 mmu which measured the cleavage of the carbon-sulfur bond of acetyl-coenzyme A in the presence of oxalacetate. Comparison of the specific activities of crude cell-free extracts indicated a much more extensive occurrence of this reaction in yeast than in A. suboxydans.
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PMID:Biosynthesis of alpha-isopropylmalic and citric acids in Acetobacter suboxydans. 603 58

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

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

Citrate synthase and malate dehydrogenase bind to mitochondrial membrane preparations obtained from various species of animals and lemon fruit. The amount of enzyme bound per mg mitochondrial protein was comparable in all tissues studied. The effect of various substrates, products, and substrate analogs on citrate synthase binding to rat liver mitochondrial inner membrane was examined. OAA was the most effective inhibitor of binding followed by AcCoA , CoA, citrate, ATP, and MgATP. Neither D- nor L- malate were effective in blocking binding. The wide distribution of binding of citrate synthase and malate dehydrogenase to the inner membrane and specificity of substrate effects on the binding of citrate synthase are discussed in relation to the possible physiologic nature of these phenomena.
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PMID:Binding of citrate synthase and malate dehydrogenase to mitochondrial inner membranes: tissue distribution and metabolite effects. 673 28

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


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