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)

The chemical nature of the inactivation of citrate synthase by S-(4-bromo-2,3-dioxobutyl)-CoA, an active site-directed irreversible inhibitor, has been investigated. Active site-directed inactivation leads to derivatization of either Lys22 by epsilon-amino Schiff base formation or Glu363 by apparent alkylation of the gamma-carboxyl group, respectively. Lys22 is labeled in the tight (catalytic) form of the enzyme while Glu363 is labeled in the open (product release) form. Glu363 and Lys22 are both located at or near the entrance to an active site in the crystal structure of citrate synthase (Remington, S., Wiegand, G., and Huber, R. (1982) J. Mol. Biol. 158, 111-152). Glu363 is in the sequence of the protomer forming the active site while Lys22 is in the sequence of the other polypeptide in the homodimer. Labeling in this region appears to inactivate the enzyme by preventing access of substrates to the active site. A distinct and separate labeling process involves derivatization of Asn192 in the tight (catalytic) form and Ser198 and/or Ser199 in the open (product release) form at a locus far removed from the active site. Labeling at the second site may simply identify chemically reactive residues, or it may identify the binding site for long chain acyl-CoA, which has been identified as a possible allosteric negative effector of citrate synthase (Caggiano, A. V., and Powell, G. L. (1979) J. Biol. Chem. 254, 2800-2806). This second labeling process apparently inactivates the enzyme by interfering with catalytically essential conformational changes.
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PMID:S-(4-bromo-2,3-dioxobutyl)-CoA labels two distinct sites on citrate synthase. 372 59

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

New techniques are needed to evaluate the luteal phase endometrium in the infertility patient. Toward this goal, we have applied quantitative microbiochemical techniques to the determination of enzyme activities in microdissected samples of each endometrial cell type (i.e., epithelial and stromal) from individual endometrial biopsies. Preliminary data on eight control patients are presented to establish a normal range of epithelial and stromal lactate dehydrogenase (LDH), beta-hydroxyacyl-CoA-dehydrogenase (beta-OH-acyl-CoA-DH), and citrate synthase (CS) enzyme activities. Significantly higher enzyme activities were found in the epithelial cell type, compared with the stromal cell type for all three enzymes. Mean enzyme activities for the eight patients, comparing epithelial to stromal values, respectively, were (1) LDH, 39.9 versus 15.4; (2) CS, 4.0 versus 1.2; and (3) beta-OH-CoA-DH, 3.9 versus 2.3 mol/kgD/hour. Enzyme activities are expressed as moles of substrate reacting per kilogram dry weight per hour (mol/kgD/hour).
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PMID:Quantitative histochemistry of late luteal phase human endometrium. 379 77

Muscle fiber distribution and muscle enzyme activity (m. vastus lat.) were investigated in 10 elite sprint cyclists and 12 nonathletes. The ratio of fast to slow muscle fibers was 2:3 in cyclists and 3:2 in nonathletes. The mean diameter of each muscle fiber type was significantly higher in the athletes. The mean enzyme activity values in mu kat X g-1 w.w. for cyclists and nonathletes, respectively, were as follows: triosephosphate dehydrogenase (TPDH), 6.2 and 3.78; lactate dehydrogenase (LDH), 4.4 and 4.59; citrate synthase (CS), 0.154 and 0.13; hydroxyacyl-CoA dehydrogenase (HAD), 0.041 and 0.07. The mean difference between groups in TPDH and in (TPDH + LDH)/(CS + HAD) ratio were statistically significant. Maximum voluntary isometric strength (knee extension) was about 17% greater in cyclists than the mean value for Czechoslovakian men of the same age. A strong positive correlation (r = 0.72) between the percent of fast glycolytic fibers (type II B) and isometric strength was observed in the cyclists. Furthermore, mean weight-compensated maximal oxygen consumption (VO2 max, ml X kg-1 X min-1) for all subjects (n = 22) was significantly related to percent of slow oxidative fibers (type I) (r = 0.75) and to the mean diameter of type II B (r = 0.58), fast oxidative-glycolytic fibers (type II A) (r = 0.68) and type I fibers (r = 0.59).
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PMID:Skeletal muscle characteristics of sprint cyclists and nonathletes. 379 40

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

The non-Michaelis-Menten kinetics, burst and steady-state periods, expressed by citrate synthase in the presence of citryl-CoA, were investigated by labelling experiments with trace amounts of [14C]acetyl-CoA. The results indicate that citrate becomes labelled in the reaction of liberated acetyl-CoA with the binary synthase.oxaloacetate complex that is transiently generated in the lyase reaction of citryl-CoA. Mediated by the hydrolase function of synthase, the counteracting citryl-CoA lyase and ligase reactions operate towards a transient flow equilibrium. This precedes the thermodynamic equilibrium and is established during the burst period; it is maintained under steady-state conditions and corresponds to the formation of transiently nonproductive synthase. The rates of both synthase partial reactions, therefore, are likewise affected. Oxaloacetate in the presence of acetyl-CoA competitively inhibits the hydrolysis of citryl-CoA and vice versa. In the synthase dependence of the burst periods and during the time dependence of the steady-state periods, nonproportionally more of physiological substrates participate in citrate formation. The nonproportional increase is a consequence of the continuously changing conditions to establish or to maintain the flow equilibrium, respectively, during the reaction progress. Third rate periods after the steady state result if the equilibrium conditions cannot be satisfied. High concentrations of oxaloacetate inhibit the expression of non-Michaelis-Menten kinetics by formation of nonproductive synthase.oxaloacetate complex. The supply of acetyl-CoA is then sufficient and the formation of the flow equilibrium prevented. The implication of the results with structural work is discussed.
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PMID:Hysteretic behaviour of citrate synthase. The reaction mechanism and the exclusion of synthase being a hysteretic enzyme. 383 Jan 75

Freezing of bovine, calf, and porcine skeletal muscles at -20 degrees C before or after rigor mortis and thawing at room temperature did not cause significant changes in the total activities of the mitochondrial enzymes lipoamide dehydrogenase, citrate synthase, and beta-hydroxyacyl-CoA-dehydrogenase. Freezing (pre or post rigor) and thawing result in a partial release of these enzymes from their binding to the inner membrane of the mitochondria. The transfer of enzyme activity into the sarcoplasmic fluid is due to damage of the mitochondrial membranes by freezing and thawing of the muscle tissue. During longer storage of the muscle at +2 degrees C the mitochondria become more labile towards freezing which may be recognised from an increased release of the enzymes (particularly in porcine muscle). Repeated freeze/thaw cycles cause an increase in the release of the three enzymes.
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PMID:[Lipoamide dehydrogenase, citrate synthase and beta-hydroxyacyl-CoA-dehydrogenase in skeletal muscles. VI. Influence of freezing (-20 degrees C) and thawing of bovine, calf and porcine muscle on activity and subcellular distribution]. 384 Mar 10

Freezing at -20 degrees C and subsequent thawing of muscles from sheep, hare and deer, and of breast and leg muscle from chicken and duck, did not result in significant changes of the total activity of the enzymes citrate synthase and beta-hydroxy-acyl-CoA-dehydrogenase; lipoamide dehydrogenase seemed to be somewhat more labile. Freezing and thawing of muscle tissue caused a partial release of these three mitochondrial enzymes into the sarcoplasmic fluid which indicates similar freeze-damage of the inner membrane of the mitochondria to that observed with bovine and porcine muscles.
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PMID:[Lipoamide dehydrogenase, citrate synthase and beta-hydroxyacyl-CoA-dehydrogenase in skeletal muscles. VII. The influence of freezing (-20 degrees C) and thawing of muscles from sheep, game and poultry on activity and subcellular distribution]. 384 Mar 11

Samples of bovine muscle (post rigor) were frozen at different temperatures between -5 degrees and -196 degrees C at different freezing rates, and thawed at room temperature. The activities of the mitochondrial enzymes lipoamide dehydrogenase, citrate synthase and beta-hydroxyacyl-CoA-dehydrogenase were determined in the supernatant of the tissue homogenates in phosphate buffer (total enzyme activity), as well as in the press juice of the intact tissue (enzyme activity in the sarcoplasma). Neither the temperature nor the rate of freezing (varying from 25.5 to 0.01 min/degrees C) showed a significant influence on the total enzyme activities. Freezing at -5 degrees and -10 degrees C (at different rates but without intracellular freezing) and thawing did not result in an appreciable release of enzymes. Below -10 degrees C the release of the three enzymes from their binding to the inner membrane of the mitochondrion into the sarcoplasmic fluid increased upon rapid freezing with decreasing temperature i.e. with increasing intracellular ice formation, whereas at slow freezing (with extracellular ice formation only) freezing below -20 degrees C did not cause further enzyme release. At freezing temperatures below -20 degrees C rapid freezing resulted in a significantly stronger release of the three enzymes than slow freezing. From these results it was concluded that the damage to mitochondrial membranes upon fast freezing is primarily a result of intracellular (and perhaps also intramitochondrial) ice formation, whereas the membrane damage during slow freezing is primarily due to dehydration caused by the migration of water from the muscle fibers into the extracellular space as a result of osmotic effects. Ion concentration in the nonfreezing fraction of tissue water seems to be only of minor importance for the disintegration of mitochondrial membranes.
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PMID:[Lipoamide dehydrogenase, citrate synthase and beta-hydroxyacyl-CoA-dehydrogenase in skeletal muscle. VIII. The influence of temperature and rate of freezing of bovine muscle on the activity and subcellular distribution of the enzymes in the thawed tissue]. 384 Mar 12

Samples of bovine muscle (post rigor) were frozen at -30 degrees C at two different rates (1.27 min/degrees C and 13.10 min/degrees C) and thawed at different rates between 1.6 (22 degrees C) and 430 min/degrees C (0 degrees C). The activities of the mitochondrial enzymes lipoamide dehydrogenase, citrate synthase, and beta-hydroxyacyl-CoA-dehydrogenase were determined in the supernatant of the tissue homogenate in phosphate buffer (total activity) and in the press juice of the intact tissue (activity in the sarcoplasma). The rate of thawing did not show a significant influence on total enzyme activities. In most cases, however, slow thawing caused a greater release of the enzymes from the mitochondria into the sarcoplasmic fluid than fast thawing, this effect being apparently independent of the rate of freezing. The greater damage to mitochondrial membranes upon slow thawing cannot be due to a longer exposure of the muscle cell to increased ionic strength in the non-freezable part of the cell water at the "critical" temperature around -3 degrees C because freezing of muscle samples at -3 degrees C and incubating them at -3 degrees C for five days resulted neither in changes of the total enzyme activities nor in a release of the three mitochondrial enzymes. From these results it is concluded that the influence of thawing rate on the damage to muscle mitochondria is probably not due to ionic effects or to recrystallization phenomena in the ice phase.
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PMID:[Lipoamide dehydrogenase, citrate synthase and beta-hydroxyacyl-CoA-dehydrogenase in skeletal muscle. IX. The influence of the rate of thawing on activity and subcellular distribution in fast and slow frozen bovine muscle]. 384 Sep 38


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