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)

Relationship of citrate synthase (EC 4.1.3.7) to the biosynthesis of glutamic acid was investigated by characterizing a new glutamic acid auxotroph FL100-D1 (glu 3) of Saccharomyces cerevisiae. Nutritional requirement of the mutant was satisfied by L-glutamic acid, L-glutamic acid peptide as well as several analogs of glutamic acid, but not by proline, ornithine, arginine, lysine or aspartic acid. The mutant was unable to utilize nonfermentable carbon sources, glycerol, acetate or lactate. Mutant glu3 unlike aconitaseless glutamic acid auxotroph glu 1, failed to accumulate 14C-citric acid in vivo from 1-14C-sodium acetate or U-14C-glutamic acid. Both spectrophotometric and radioactive assay procedures demonstrated a lack of significant citrate synthase activity in the dialysed extract of the mutant compared to the wild type strain. Mutant glu 3 complemented with glu 1 and glu 2 individually in vivo and exhibited a significant aconitase (EC 4.2.1.3) activity in vitro.
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PMID:Citrate synthaseless glutamic acid auxotroph of Saccharomyces cerevisiae. 110 43

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.
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PMID:Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae. 144 89

The possible involvement of arginyl and lysyl side chains of chloramphenicol acetyltransferase (CAT) in binding coenzyme A (CoA) was studied by means of chemical modification, site-directed mutagenesis, variation in ionic strength, use of competitive inhibitors or substrate analogues, and X-ray crystallography. Unlike a number of enzymes, including citrate synthase, CAT does not employ specific ion pairs with the phosphoanionic centers of CoA to bind the acetyl donor, and arginyl residues play no role in recognition of the coenzyme. Although phenylglyoxal inactivates CAT reversibly, it does so by the formation of an unstable adduct with a thiol group, that of Cys-31 in the chloramphenicol binding site. The inhibitory effect of increasing ionic strength on kcat/Km(acetyl-CoA) can be explained by long-range electrostatic interactions between CoA and the epsilon-amino groups of Lys-54 and Lys-177, both of which are solvent-accessible. The epsilon-amino group of Lys-54 contributes 1.3 kcal.mol-1 to the binding of acetyl-CoA via interactions with both the 3'- and 5'-phosphoanions of CoA. Lys-177 contributes only 0.4 kcal.mol-1 to the productive binding of acetyl-CoA, mediated by long-range (approximately 14 A) interactions with the 5'-alpha- and -beta-phosphoanions of CoA. The combined energetic contribution of Lys-54 and Lys-177 to acetyl-CoA binding (1.7 kcal.mol-1) is less than that previously demonstrated (2.4 kcal.mol-1) for a simple hydrophobic interaction between Tyr-178 and the adenine ring of CoA (Day & Shaw, 1992).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Acetyl coenzyme A binding by chloramphenicol acetyltransferase: long-range electrostatic determinants of coenzyme A recognition. 156 67

A cDNA that encodes pig citrate synthase (PCS) was inserted into a plasmid T7 vector and was expressed in an E. coli gltA mutant. Up to 10 mg of purified PCS was obtained from 2 liters of E. coli. The mammalian protein produced in E. coli comigrated with the enzyme purified from pig heart on a SDS-polyacrylamide gel (SDS-PAGE) with an Mr of 50,000, and reacted with a polyclonal antibody directed against pig heart citrate synthase. The Vmax and Km of the expressed PCS were indistinguishable from those of the pig heart enzyme. The PCS produced in E. coli did not contain the trimethylation modification of Lys 368, characteristic of the pig heart enzyme. These data suggest that the PCS protein produced in E. coli is catalytically similar to the enzyme purified from pig heart and methylation of Lys 368 is not essential for catalysis.
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PMID:Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from Escherichia coli. 314 69

The description of hysteretic behaviour of citrate synthase is completed with the demonstration of a burst period in the citryl-CoA lyase reaction. The kinetics of this partial reaction show symmetry to those of the citryl-CoA hydrolase reaction. The amplitudes of the burst periods of each partial reaction are proportional to synthase activity. Using the synthase species proteolytically nicked by endoproteinase Lys-C, a standard was elaborated to determine the actual ratio of hydrolase over lyase reactions which was found to be 0.72:0.28. The ratio found with native synthase averaged 0.8:0.2. These and other results indicate that less oxaloacetate is liberated from the synthase than is actually generated in the lyase reaction of citryl-CoA. The temperature dependence of hysteretic behaviour of both partial reactions is consistent with the participation of citryl-CoA-derived physiological substrates in the generation of this behaviour. More hydrolytic products were formed at low than at high temperature. As shown with the proteolytically nicked synthase species indicated above, this effect is related to different temperature coefficients of the partial reactions. The apparent activation energies of the citryl-CoA hydrolase and lyase reactions, 26.7 kJ X mol-1 and 44.6 kJ X mol-1, respectively, were determined. The action of established synthase inhibitors on the expression of hysteretic behaviour is described and discussed.
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PMID:Hysteretic behaviour of citrate synthase. Symmetry in the kinetics of the synthase partial reactions. 383 Jan 63

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

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

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

Rat kidney homogenates metabolize N6-trimethyl-lysine to N-trimethylammoniobutyrate, but not to carnitine. The first step in this conversion is the hydroxylation of trimethyl-lysine to form 3-hydroxy-N6-trimethyl-lysine. An assay system was developed in which hydroxylation of trimethyl-lysine is linear with respect to both time and homogenate protein concentration. The rate is 5 nmol of 3-hydroxy-N6-trimethyl-lysine formed/min per mg of homogenate protein. The cofactors required are ascorbate, alpha-oxoglutarate, FeSO4, and O2. Catalase and dithiothreitol give a 20% stimulation. Ca2+ produces a 2-fold increase in specific activity and cannot be replaced by Mg2+, Mn2+ or Zn2+. These last three bivalent cations lead to a decreased activity. Subcellular distribution studies demonstrate that trimethyl-lysine hydroxylase activity parallels the distribution profile of succinate dehydrogenase and citrate synthase. Thus trimethyl-lysine hydroxylase has a mitochondrial localization. Distribution of trimethyl-lysine hydroxylase activity between cortex and medulla of kidney if 67 and 33% respectively, similar to mitochondrial distribution.
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PMID:Carnitine biosynthesis. Hydroxylation of N6-trimethyl-lysine to 3-hydroxy-N6-trimethyl-lysine. 677 70

We analyzed mitochondrial DNA (mtDNA) from 7 patients in four families with adult onset limb-girdle type mitochondrial myopathy to clarify their genetic background. The patients, 2 men and 5 women, showed common clinical features, characterized by isolated skeletal myopathy, high serum creatine kinase level, ragged-red fibers and cytochrome c oxidase-defective fibers. Analysis of muscle biopsy specimens indicated that cytochrome c oxidase activity was decreased relative to that of citrate synthase in 5 of the 7 patients. Southern blotting and direct sequence analyses showed an A-to-G homoplasmic transition at np8291 and intergenic COII/tRNA (Lys) 9bp deletion in all patients. This substitution was detected in only 2 of 600 control individuals including healthy subjects and patients with other neuromuscular disorders; these 2 individuals had diabetes mellitus and myotonic dystrophy, respectively. Consequently, the mtDNA transition at np8291 was a rare polymorphism. However, the 7 patients we studied had identical clinical, pathological, biochemical, and genetic features. Therefore, limb-girdle type mitochondrial myopathy with this rare polymorphism may form a subgroup of adult onset mitochondrial myopathy.
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PMID:Adult onset limb-girdle type mitochondrial myopathy with a mitochondrial DNA np8291 A-to-G substitution. 1031 90


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