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)

A rapid and simple spectrophotometric method was developed to measure the activity of the condensing enzyme component of the microsomal fatty acid chain elongation system. The intermediate product of the condensation reaction is the beta-ketoacyl CoA which exists in two tautomeric forms, i.e., keto and enol. The addition of bovine serum albumin (BSA) to a cuvette cell containing a beta-ketoacyl CoA derivative resulted in the formation of a 303-nm absorbance peak, characteristic of enolate formation. The beta-ketoacyl CoAs with carbon chain length of 6 to 18 interacted with BSA to produce the 303-nm peak; acetoacetyl CoA was the only beta-keto compound tested which did not interact with BSA to produce the peak. Other compounds which were unaffected by BSA included CoA, free beta-keto acid, beta-hydroxyacyl CoA, acyl CoA, trans-2-enoyl CoA, and malonyl CoA. BSA could not be replaced by ovalbumin; furthermore, denatured (boiling) BSA could not induce the 303-nm peak. The specific activity of the condensing enzyme measured by the spectrophotometric method compares favorably with the activity obtained by the radioactive method. The apparent extinction coefficient (epsilon) for the absorbance peak generated by the beta-keto thioester varied from 5 to 30 mM-1 cm-1 depending on the beta-keto derivative. The spectrophotometric procedure can be used in the determination of the condensing enzyme activity in not only hepatic microsomes but also in kidney and brain microsomes both of which have significantly lower activity. The advantages of the novel method over the radioactive method are that (i) it does not involve the use of radioactive compounds, (ii) it is much less cumbersome and significantly less costly, and (iii) it is rapid and easy to perform.
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PMID:Spectrophotometric assay for the condensing enzyme activity of the microsomal fatty acid chain elongation system. 277 74

The purified carbon monoxide dehydrogenase from Clostridium thermoaceticum is the only protein required to catalyze an exchange reaction between carbon monoxide and the carbonyl group of acetyl-CoA. This exchange requires that the CO dehydrogenase bind the methyl, the carbonyl, and the CoA groups of acetyl-CoA, then equilibrate the carbonyl with CO in the solution and re-form acetyl-CoA. CoA is not necessary for the exchange and, in fact, inhibits the reaction. These studies support the view that CO dehydrogenase is the condensing enzyme that forms acetyl-CoA from its component parts. Carbon dioxide also exchanges with the C-1 of acetyl-CoA, but at a much lower rate than does CO. At 50 degrees C and pH 5.3, the optimal pH, the turnover number is 70 mol of CO exchanged per min/mol of enzyme. Low potential electron carriers are stimulatory. The Km app for stimulation by ferredoxin is 50-fold less than the value for flavodoxin. Neither ATP or Pi stimulate the exchange. The EPR spectrum of the CO-reacted enzyme is markedly changed by binding of CoA or acetyl-CoA. Arginine residues of the CO dehydrogenase appear to be involved in the active site, possibly by binding acetyl-CoA. Mersalyl acid, methyl iodide, 5,5-dithiobis-(2-nitrobenzoate), and sodium dithionite inhibit the exchange reaction. A scheme is presented to account for the role of CO dehydrogenase in the exchange reaction and in the synthesis of acetate.
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PMID:Acetate biosynthesis by acetogenic bacteria. Evidence that carbon monoxide dehydrogenase is the condensing enzyme that catalyzes the final steps of the synthesis. 298 90

The first condensation reaction in the fatty acid biosynthetic pathway in Escherichia coli was rate-limiting as judged by analysis of the relative pool sizes of acyl carrier protein (ACP) thioester intermediates in vivo. Comparable concentrations of acetyl-ACP, malonyl-ACP, and nonesterified ACP were present during logarithmic growth, whereas long-chain acyl-ACP comprised a minor fraction of the total ACP pool. The antibiotic cerulenin was used to irreversibly inhibit both beta-ketoacyl-ACP synthases I and II. However, acyl-ACP formation in vivo was not blocked by this antibiotic, and short-chain (4-8-carbon) acyl-ACPs increased to 60% of the total ACP pool in cerulenin-treated cells. These data suggested that existence of a cerulenin-resistant condensing enzyme that was capable of catalyzing the initial steps in chain elongation. A unique enzymatic activity, acetoacetyl-ACP synthase, that specifically catalyzed the condensation of malonyl-ACP and acetyl-ACP was detected in E. coli cell extracts. Acetoacetyl-ACP synthase activity was not inhibited by cerulenin and was present in extracts prepared from a double mutant harboring genetic lesions in beta-ketoacyl-ACP synthases I and II (fabB20 fabF3). These data point to the condensation of malonyl-ACP and acetyl-ACP as the rate-controlling reaction in fatty acid biosynthesis and implicate acetoacetyl-ACP synthase as the pacemaker of fatty acid production in organisms and organelles that possess dissociated (Type II) fatty acid synthase systems.
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PMID:Acetoacetyl-acyl carrier protein synthase, a potential regulator of fatty acid biosynthesis in bacteria. 329 37

The feeding of 2% di(2-ethylhexyl)phthalate (DEHP) to rats increased the hepatic microsomal elongation of palmitoyl-CoA by about twofold, while those of palmitoleoyl-CoA and gamma-linolenoyl-CoA decreased to 83 and 63%, respectively, of the control values. When component reactions of the elongation pathway were measured, it was observed that only the activity of condensing enzyme was increased by twofold, while those of beta-ketostearoyl-CoA reductase, beta-hydroxypalmitoyl-CoA dehydrase, and trans-2-hexadecenoyl-CoA reductases were not affected. Furthermore, the time course for induction of both condensation and elongation of palmitoyl-CoA was similar. In vitro addition of DEHP had no effect on either condensation or elongation. Thus, these results indicate that the peroxisomal proliferator induces only the condensing enzyme which is the regulatory and rate-limiting step of elongation sequence. The DEHP treatment also markedly enhanced the cytosolic NADPH-generating activities of glucose-6-PO4 dehydrogenase (2.2-fold) and malic enzyme (7.3-fold). Unexpectedly, the activities of fatty acid synthetase and citrate cleavage enzyme were unaffected. These results are discussed in light of the fact that these lipogenic enzymes are coordinately induced by diet or hormones.
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PMID:Effect of the peroxisomal proliferator di(2-ethylhexyl)phthalate on component reactions of the rat hepatic microsomal fatty acid chain elongation system and on other hepatic lipogenic enzymes. 352 64

Prodigiosin condensing enzyme (PCE) activities were present in Serratia marcescens wild type 08, mutants OF, WF and 9-3-3. Their specific activities exhibited different maxima and at different times during the late log phase or the early stationary phase of cell growth. The levels of prodigiosin and its precursors also showed a significant increase at this period. The results support that prodigiosin and/or its precursors are secondary metabolites. The ubiquity of the PCE activity in mutants deficient in prodigiosin biosynthesis suggest that this particular enzyme may also be present in non-pigmented clinical isolates.
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PMID:Relationship of prodigiosin condensing enzyme activity to the biosynthesis of prodigiosin and its precursors in Serratia marcescens. 355 18

The present study examines the effect of the acetylenic thioester dec-2-ynoyl-CoA (delta 2 10 identical to 1-CoA) on the microsomal fatty acid chain elongation pathway in rat liver. When the individual reactions of the elongation system were measured in the presence of delta 2 10 identical to 1-CoA, the trans-2-enoyl-CoA reductase activity was markedly inhibited (Ki = 2.5 microM), whereas the activities of the condensing enzyme, the beta-ketoacyl-CoA reductase, and the beta-hydroxyacyl-CoA dehydrase were not affected. The absence of inhibition of total microsomal fatty acid elongation was attributed to the significant accumulation of the intermediates, beta-hydroxyacyl-CoA and trans-2-enoyl-CoA, without formation of the saturated elongated product, indicating that the trans-2-enoyl-CoA reductase-catalyzed reaction was the only site affected by the inhibitor. The nature of the inhibition was noncompetitive. In contrast to the delta 2 10 identical to 1-CoA, delta 3 10 identical to 1-CoA did not inhibit trans-2-enoyl-CoA reductase activity, suggesting that the mode of inhibition was not via formation of the 2,3-allene derivative. Based on the observation (a) that p-chloromercuribenzoate markedly inhibits reductase activity, (b) that dithiothreitol protects the enzyme against inactivation by delta 2 10 identical to 1-CoA, (c) of the spectral manifestation of the interaction between thiol reagents and delta 2 10 identical to 1-CoA depicting an absorbance peak similar to that of the beta-ketoacyl thioester-Mg2+ enolate complex, (d) of a similar absorbance spectrum formed by the interaction between delta 2 10 identical to 1-CoA and liver microsomes, and (e) of the absence of formation of a similar spectrum by delta 3 10 identical to 1-CoA, trans-2-10:1-CoA, or delta 2 10 identical to 1 free acid with liver microsomes, we propose that delta 2 10 identical to 1-CoA inactivates trans-2-enoyl-CoA reductase by covalently binding to a critical sulfhydryl group at or in close proximity to the active site of the enzyme.
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PMID:Site of inhibition of rat liver microsomal fatty acid chain elongation system by dec-2-ynoyl coenzyme A. Possible mechanism of inhibition. 375 85

Baker's yeast fermenting on D-glucose converts 2-substituted C6-C3 alpha,beta-unsaturated aromatic aldehydes into the corresponding 3-phenylprop-2-en-1-ols and 3-phenylpropan-1-ols, and into the 4-substituted (2S,3R)-5-phenylpent-4-en-2,3-ols. The formation of the C6-C3 alcohols from the aldehydes by baker's yeast was already known, but the production of the methyl diols is new. The conversion of C6-C3 alpha,beta-unsaturated aldehydes into the C6-C5 methyl diols can be viewed as the overall consequence of two distinct chemical operations: (1) addition of a C2 unit equivalent to acetaldehyde onto the Si-face of the carbonyl carbon of the unsaturated aldehyde forms the (R)-alpha-hydroxy ketone in an acyloin-type condensation, and (2) reduction of this intermediate on the Re-face of the carbonyl gives the diol actually isolated. There is some tolerance by the enzymic system(s) involved in the reaction(s) leading from the C6-C3 alpha,beta-unsaturated aromatic aldehydes to the 4-substituted (2S,3R)-5-phenylpent-4-en-2,3-ols as far as the structure of the aromatic aldehydes and the substitutents in the alpha position are concerned, but acetaldehyde is the only aldehyde accepted as second terminus of the reaction. However, synthetic alpha-hydroxy ketones, prepared from aldehydes that cannot be directly converted by yeast into the corresponding methyl diols, are reduced by yeast. This indicates that the reason direct conversion of the aldehydes does not occur is that these materials probably cannot be accepted as substrates by the condensing enzyme(s). The (2S,3R)-diols can be used instead of natural carbohydrates as starting materials for the synthesis of optically active forms of natural products belonging to different structural classes. Applications of these diols in the synthesis of L-daunosamine, the natural form of vitamin E and other products are discussed.
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PMID:Stereochemistry and synthetic applications of products of fermentation of alpha,beta-unsaturated aromatic aldehydes by baker's yeast. 389 37

Spheroplast membranes (spheroplast envelopes) of strain 2091 of group B Neisseria meningitidis were prepared by a procedure that included lysozyme treatment of the cells and osmotic lysis of the resulting spheroplasts. Electron microscopy revealed that the membranes consisted of two unit layers, generally parallel to each other. The membrane preparation migrated as a single component in a 40 to 70% sucrose gradient and consisted of 62% protein, 28% lipid, 9% ribonucleic acid, small amounts of carbohydrate, hexosamine, and deoxyribonucleic acid. When 1 or 10 mug (dry weight) was injected intravenously into rabbits, a mild pyrogenic reaction was elicited. In immunodiffusion tests, immune rabbit serum prepared against spheroplast membranes produced three major precipitin lines, with the homologous antigen solubilized with sodium dodecyl sulfate, and a single line with untreated antigen. The immune serum also reacted with a cell wall antigen, and to a lesser extent with some of the cytoplasmic antigens. Succinate dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH) oxidase activities were found to be associated with the spheroplast membranes. NADH dehydrogenase also was associated with the membranes but was gradually released and recovered in other fractions. Glutamate-oxaloacetate transaminase, glutamate, glucose-6-phosphate, and isocitrate dehydrogenase activities were not found in the membrane preparation. About one-third of these enzymatic activities were recovered in the supernatant fluid after the sedimentation of the spheroplasts and two-thirds were recovered in the cytoplasmic fraction. N-acetylneuraminic acid (NAN)-condensing enzyme and cytidine monophosphate-NAN synthesizing enzyme also were identified in this organism. These enzymes were not associated with the membranes and were recovered from extracts from whole cells, spheroplasts, or cells exposed to osmotic shock, as well as from spheroplast supernatant and shock fluids. It is concluded that the spheroplast membranes of the strain of meningococci used in these studies are typical of those recovered from gram-negative bacteria.
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PMID:Characterization of spheroplast membranes of Neisseria meningitidis group B. 463 Jul 22

Various acyl-acyl carrier protein intermediates in saturated and unsaturated fatty acid biosynthesis were tested as substrates for beta-ketoacyl-acyl carrier protein synthetase. With both classes of substrates the condensing enzyme in fatty biosynthesis demonstrates specificities which indicate that it might be an important factor in determining fatty acid chain length in Escherichia coli.
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PMID:Enzyme specificity as a factor in regulation of fatty acid chain length in Escherichia coli. 492 Jun 54

The isolation and biochemical characterization of a Saccharomyces cerevisiae mutant, which grows only when emulsified myristic, palmitic, stearic, or oleic acid is added to the growth medium, is described. The mutant contains an enzymatically inactive fatty acid synthetase complex. The gene affected, preliminarily designated by the symbol fas, exhibits a 2:2 nuclear inheritance pattern. The formation of fatty acid synthetase in yeast is constitutive and not subject to repression by long chain fatty acids. After extensive purification, the mutant fatty acid synthetase was obtained as an essentially homogeneous protein with a sedimentation constant identical to that of the wild type enzyme. A systematic study of the seven reaction steps involved in fatty acid biosynthesis revealed that the enzyme catalyzing the condensation of acetate and malonate to acetoacetate was completely inactive in the mutant. The other six component enzymes had identical specific activities in the mutant and in the wild type fatty acid synthetase complexes. It is concluded that the mutant described harbors a missense mutation in the structural genes of either the condensing enzyme of the "acyl carrier protein" component of the fatty acid synthetase complex.
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PMID:A Saccharomyces cerevisiae mutant defective in saturated fatty acid biosynthesis. 494 77


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