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)

The alpha-ketoglutarate dehydrogenase complex of either pig heart or Escherichia coli catalyzes a NAD- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate which is stereoselective toward the L-isomer of this hydroxyketo acid. L-Malyl-CoA is the product of the reaction; the evidence includes observing (a) a steady increase in absorbance at 230 nm during the oxidation of 2-keto-4-hydroxyglutarate, (b) a positive response of oxidation reaction mixtures to neutral hydroxylamine, (c) loss of the two foregoing results concomitant with release of thiol-reacting material and the formation of free malate when reaction mixtures are heated, (d) formation of a hydroxamate which has chromatographic mobilities identical to that of chemically synthesized malate hydroxamate, (e) enzymatic formation of a radioactive product from 14C-labeled 2-keto-4-hydroxyglutarate which co-migrates with chemically synthesized malyl-CoA, and (f) hydrolysis of the product by citrate synthase, an enzyme absolutely specific for citryl-CoA and L-malyl-CoA. A 1:1:1 stoichiometric relationship exists between the amount of 2-keto-4-hydroxyglutarate oxidized, NAD reduced, and malate (or malyl-CoA) formed. Results from studies in which either 14C-labeled 2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate is incubated with mixtures of purified enzymes or extracts of E. coli support the suggestion that the aldolase which preferentially catalyzes formation of L-2-keto-4-hydroxyglutarate from pyruvate plus glyoxylate in E. coli is coupled with the oxidative decarboxylation of this substrate, as reported here, and other enzymes in a multistep pyruvate-catalyzed cyclic oxidation of glyoxylate.
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PMID:Malyl-CoA formation in the NAD-, CoASH-, and alpha-ketoglutarate dehydrogenase-dependent oxidation of 2-keto-4-hydroxyglutarate. Possible coupled role of this reaction with 2-keto-4-hydroxyglutarate aldolase activity in a pyruvate-catalyzed cyclic oxidation of glyoxylate. 638 79

The detailed proof of the 437-residue amino acid sequence (Mr 48,969) of porcine heart citrate synthase (EC 4.13.7) is described. The S-carboxymethylated protein has been cleaved at methionine (cyanogen bromide) and arginine (trypsin digest of citraconylated enzyme) residues to yield 14 and 17 major peptides, respectively. Peptides were initially fractionated by gel filtration, and those useful for sequence analysis were purified by high-performance liquid chromatography. Sequence analyses were performed on these primary peptides and on subpeptides generated by cleavage with the bromine adduct of 2-[(2-nitrophenyl)sulfenyl]-3-methylindole, Staphylococcus aureus V8 protease, trypsin, chymotrypsin, or acid. The overall sequence was confirmed by analyzing products of cleavage by hydroxylamine, acid, and subtilisin. A novel feature of the sequence is the identification of trimethyllysine at residue 368.
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PMID:Complete amino acid sequence of porcine heart citrate synthase. 709 27

The assay of oxaloacetate and alpha-ketoglutarate in biological samples is complicated by their chemical instability and low concentrations. We present a quantitative assay for physiological concentrations of these metabolites by isotope dilution gas chromatography-mass spectrometry. Samples are spiked with the corresponding internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate prior to their treatment with hydroxylamine. After ethyl acetate extraction and evaporation of the organic phases, the oximes are converted to t-butyldimethylsilyl ethers and analyzed by selected ion monitoring gas chromatography-mass spectrometry of the [M-57]+ ion in electron impact. Although the internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate are not commercially available, they can easily be synthesized in 30 min by reacting [1,2,3,6-13C4]citrate with citrate lyase, and L-[U-13C5]glutamate with pyruvate and glutamate-pyruvate transaminase, respectively. Because of their chemical instability, the internal standards are prepared on the day of the analysis. A stock solution of [1,2,3,6-13C4]citrate is prepared from L-[U-13C4]aspartate using citrate synthase and glutamate-oxaloacetate transaminase and then purified and kept frozen until required. The detection limit of the method is 0.05 nmol in a given sample. The method was applied to measurements of oxaloacetate and alpha-ketoglutarate in human blood and rat liver.
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PMID:Assay of blood and tissue oxaloacetate and alpha-ketoglutarate by isotope dilution gas chromatography-mass spectrometry. 773 61

Rat carnitine palmitoyltransferase (CPT) II was expressed in Saccharomyces cerevisiae. Mitochondrial fractions prepared from the cells displayed high CPT activity and reacted with an antibody to the rat protein on immunoblots, whereas no activity or immunoreactive protein was observed in control cells. The recombinant enzyme was largely membrane associated. Treatment of the expressed protein with diethyl pyrocarbonate, a reagent that modifies histidine residues, abolished CPT activity, but this was completely restored by reversal of the modification with hydroxylamine. It is inferred that a histidine residue plays a critical role in CPT function. Expression and analysis of site-directed mutants of CPT II showed that histidine 372, as well as aspartates 376 and 464 (all conserved throughout the carnitine/choline acyltransferase family), are essential for catalytic activity. The data suggest that the mechanism by which CPT II effects transesterification between palmitoyl-CoA and carnitine possibly involves histidine 372 and one of these aspartate residues, interacting with the carnitine hydroxyl group, in a reaction analogous to that carried out by a histidine/aspartate/serine catalytic triad in certain other enzyme systems. Mutagenic analysis of a region of CPT II that is highly conserved among the carnitine and choline acyltransferases, and which is homologous to the "adenine binding loop" of citrate synthase, implies that it has no similar function in CPT II.
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PMID:Catalytically important domains of rat carnitine palmitoyltransferase II as determined by site-directed mutagenesis and chemical modification. Evidence for a critical histidine residue. 803 73