The kinetic properties of citrate synthase from rat liver mitochondria

1. Citrate synthase (EC 4.1.3.7) was purified 750-fold from rat liver. 2. Measurements of the Michaelis constants for the substrates of citrate synthase gave values of 16mum for acetyl-CoA and 2mum for oxaloacetate. Each value is independent of the concentration of the other substrate. 3. The inhibition of citrate synthase by ATP, ADP and AMP is competitive with respect to acetyl-CoA. With respect to oxaloacetate the inhibition by AMP is competitive, but the inhibition by ADP and ATP is mixed, being partially competitive. 4. At low concentrations of both substrates the inhibition by ATP is sigmoidal and a Hill plot exhibits a slope of 2.5. 5. The pH optimum of the enzyme is 8.7, and is not significantly affected by ATP. 6. Mg(2+) inhibits citrate synthase slightly, but relieves the inhibition caused by ATP in a complex manner. 7. At constant total adenine nucleotide concentration made up of various proportions of ATP, ADP and AMP, the activity of citrate synthase is governed by the concentration of the sum of the energy-rich phosphate bonds of ADP and ATP. 8. The sedimentation coefficient of the enzyme, as measured by activity sedimentation, is 6.3s, equivalent to molecular weight 95000.     

Distribution of metabolites between the cytosolic and mitochondrial compartments of hepatocytes isolated from fed rats

In isolated hepatocytes from normal fed rats, the subcellular distribution of malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH has been determined by a modified digitonin method. Incubation with various substrates (lactate, pyruvate, alanine, oleate, oleate plus lactate, ethanol and aspartate) markedly changed the total cellular amounts of metabolites, but their distribution between the cytosolic and mitochondrial compartments was kept fairly constant. In the presence of lactate, pyruvate or alanine, about 90% of cellular aspartate, malate and oxaloacetate, and 50% of citrate was located in the cytosol. The changes in acetyl-CoA in the cytosol were opposite to those in the mitochondrial space, the sum of both remaining nearly constant. The mitochondrial acetyl-CoA/CoASH ratio ranged from 0.3-0.9 and was positively correlated with the rate of ketone body formation. The mitochondrial/cytosolic (m/c) concentration gradients for malate, citrate, 2-oxoglutarate, glutamate, aspartate, oxaloacetate, acetyl-CoA and CoASH averaged from hepatocytes under different substrate conditions were determined to be 1.0, 8.8, 1.6, 2.2, 0.5, 0.7, 13 and 40, respectively. From the distribution of citrate, a pH difference of 0.3 across the inner mitochondrial membrane was calculated, yet lower values resulted from the m/c gradients of 2-oxoglutarate, glutamate and malate. The mass action ratios for citrate synthase and mitochondrial aspartate aminotransferase have been calculated from the metabolite concentrations measured in the mitochondrial pellet fraction. A comparison with the respective equilibrium constants indicates that in intact hepatocytes, neither enzyme maintains its reactants at equilibrium. On the assumption that mitochondrial malate dehydrogenase and 3-hydroxybutyrate dehydrogenase operate near equilibrium, the concentration of free oxaloacetate appears to be 0.3-2 micron, depending on the substrate used. Plotting the calculated free mitochondrial oxaloacetate concentration against the citrate concentration measured in the mitochondrial pellet yielded a hyperbolic saturation curve, from which an apparent Km of citrate synthase for oxaloacetate in the intact cells of 2 micron can be derived, which is comparable to the value determined with purified rat liver citrate synthase. The results are discussed with respect to the supply of substrates and effectors of anion carriers and of key enzymes of the tricarboxylic acid cycle and fatty acid biosynthesis.     

Identification and characterization of a re-citrate synthase in Dehalococcoides strain CBDB1

The genome annotations of all sequenced Dehalococcoides strains lack a citrate synthase, although physiological experiments have indicated that such an activity should be encoded. We here report that a Re face-specific citrate synthase is synthesized by Dehalococcoides strain CBDB1 and that this function is encoded by the gene cbdbA1708 (NCBI accession number CAI83711), previously annotated as encoding homocitrate synthase. Gene cbdbA1708 was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified. The enzyme catalyzed the condensation of oxaloacetate and acetyl coenzyme A (acetyl-CoA) to citrate. The protein did not have homocitrate synthase activity and was inhibited by citrate, and Mn2+ was needed for full activity. The stereospecificity of the heterologously expressed citrate synthase was determined by electrospray ionization liquid chromatography-mass spectrometry (ESI LC/MS). Citrate was synthesized from [2-(13)C]acetyl-CoA and oxaloacetate by the Dehalococcoides recombinant citrate synthase and then converted to acetate and malate by commercial citrate lyase plus malate dehydrogenase. The formation of unlabeled acetate and 13C-labeled malate proved the Re face-specific activity of the enzyme. Shotgun proteome analyses of cell extracts of strain CBDB1 demonstrated that cbdbA1708 is expressed in strain CBDB1.     

Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role.

The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.     

Convenient rapid determination of picomole amounts of oxaloacetate and aspartate

The assay of oxaloacetate based on the citrate synthase catalyzed conversion of labeled acetyl-CoA to citrate has been greatly simplified by the development of a charcoal separation method for the selective adsorption of acetyl-CoA. An application of this procedure for the determination of oxaloacetate in rat livers is described. By coupling to glutamate oxaloacetate transaminase, the procedure enables determination of aspartate. It allows also a sensitive assay of glutamate oxaloacetate transaminase activity.     

Interaction of ligands with pig heart citrate synthase: conformational changes and catalysis.

The fluorescence polarization of 8-hydroxypyrene (1,3,6)trisulfonate (HPT) increases upon interaction with pig heart citrate synthase. Titration of HPT with increasing concentrations of citrate synthase exhibits a hyperbolic saturation behavior, from which the dissociation constant of the enzyme-HPT complex (3.64 +/- 0.3 microM) was determined. The enzyme-HPT interaction is competitively inhibited by oxaloacetate (but not affected by acetyl CoA) with a Ki of 4.3 +/- 1.8 microM. This value is similar to the dissociation constant (Kd = 4.5 +/- 1.6 microM) for the enzyme-oxalocetate complex (determined in the absence of any effector ligand), as well as to the Km for oxaloacetate (3.9 +/- 0.7 microM) in a steady-state citrate synthase catalyzed reaction at a saturating concentration of acetyl CoA. However, the dissociation constant for the citrate synthase-oxaloacetate complex determined by the urea denaturation method is at least 25-fold lower than those determined by the other methods. This suggests an effector role of urea in strengthening the enzyme-oxaloacetate interaction. At low nondenaturing concentrations, urea inhibits the citrate synthase catalyzed reaction in an uncompetitive manner with respect to oxaloacetate, i.e., the Km for oxaloacetate decreases with an increase in urea concentration. This further suggests that urea stabilizes the interaction between citrate synthase and oxaloacetate. The effect of urea is specific for the substrate oxaloacetate, and not for the substrate analogue, HPT, although both these ligands bind citrate synthase with equal affinities, and protect the enzyme against thermal denaturation with equal magnitudes. The results presented herein are discussed in the light of known conformational states of the enzyme.     

Changes in the contents of adenine nucleotides and intermediates of glycolysis and citric acid cycle in flight muscle of the locust upon flight and their relationship to the control of the cycle

1. The contents of some intermediates of glycolysis, the citric acid cycle and adenine nucleotides have been measured in the freeze-clamped locust flight muscle at rest and after 10s and 3min flight. The contents of glucose 6-phosphate, pyruvate, alanine and especially fructose bisphosphate and triose phosphates increased markedly upon flight. The content of acetyl-CoA is decreased after 3min flight whereas that of acetylcarnitine is decreased markedly after 10s flight, but returns towards the resting value after 3min flight. The content of citrate is markedly decreased after both 10s and 3min flight, whereas that of isocitrate is changed very little after 10s and is increased by 50% after 3min. The content of oxaloacetate is very low in insect flight muscle and hence it was measured by a sensitive radiochemical assay. The content of oxaloacetate increased about 2-fold after 3min flight. A similar change was observed in the content of malate. The content of ATP decreased about 15%, whereas those of ADP and AMP increased about 2-fold after 3min flight. 2. Calculations based on O(2) uptake of the intact insect indicate that the rate of the citric acid cycle must be increased >100-fold during flight. Consequently, if citrate synthase catalyses a non-equilibrium reaction, the activity of the enzyme must increase >100-fold during flight. However, changes in the concentrations of possible regulators of citrate synthase, oxaloacetate, acetyl-CoA and citrate (which is an allosteric inhibitor), are not sufficient to account for this change in activity. It is concluded that there may be much larger changes in the free concentration of oxaloacetate than are indicated by the changes in the total content of this metabolite or that other unknown factors must play an additional role in the regulation of citrate synthase activity. 3. The increased content of oxaloacetate could be produced via pyruvate carboxylase, which may be stimulated during the early stages of flight by the increased concentration of pyruvate. 4. The decreases in the concentrations of citrate and alpha-oxoglutarate indicate that isocitrate dehydrogenase and oxoglutarate dehydrogenase may be stimulated by factors other than their pathway substrates during the early stages of flight. 5. Calculated mitochondrial and cytosolic NAD(+)/NADH ratios are both increased upon flight. The change in the mitochondrial ratio indicates the importance of the intramitochondrial ATP/ADP concentration ratio in the regulation of the rate of electron transfer in this muscle.     

Activation by spermine of citrate synthase from porcine heart

Spermine activated citrate synthase from porcine heart by decreasing the Km value for the substrate oxaloacetate without affecting the maximal velocity. Spermine markedly increased the maximal velocity of the saturation function with respect to acetyl-CoA as the substrate under conditions of intracellular concentrations of oxaloacetate, but the enzyme was not activated by spermine under conditions of higher concentrations of oxaloacetate. The concentration of spermine required for 50% activation of the enzyme was about 50 microM. Spermidine showed only a little activation, while putrescine caused no activation. Spermine, which contributes to an activation of Ca2(+)-sensitive dehydrogenases of the citric acid cycle by enhancing Ca2+ uptake into mitochondria, can activate citrate synthase directly, and is responsible for the stimulation of oxidative metabolism in mitochondria.     

Improving biobutanol production in engineered Saccharomyces cerevisiae by manipulation of acetyl-CoA metabolism

Recently, butanols (1-butanol, 2-butanol and iso-butanol) have generated attention as alternative gasoline additives. Butanols have several properties favorable in comparison to ethanol, and strong interest therefore exists in the reconstruction of the 1-butanol pathway in commonly used industrial microorganisms. In the present study, the biosynthetic pathway for 1-butanol production was reconstructed in the yeast Saccharomyces cerevisiae. In addition to introducing heterologous enzymes for butanol production, we engineered yeast to have increased flux toward cytosolic acetyl-CoA, the precursor metabolite for 1-butanol biosynthesis. This was done through introduction of a plasmid-containing genes for alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA synthetase (ACS), and acetyl-CoA acetyltransferase (ERG10), as well as the use of strains containing deletions in the malate synthase (MLS1) or citrate synthase (CIT2) genes. Our results show a trend to increased butanol production in strains engineered for increased cytosolic acetyl-CoA levels, with the best-producing strains having maximal butanol titers of 16.3 mg/l. This represents a 6.5-fold improvement in butanol titers compared to previous values reported for yeast and demonstrates the importance of an improved cytosolic acetyl-CoA supply for heterologous butanol production by this organism.     

Factors regulating gluconeogenesis in chick embryo liver]

The ratio NAD+/NADH in cytoplasm and mitochondria of chicken embryo liver does not change up to the stage of hatching. After the hatching this ratio decreases 2-fold in both cytoplasm and mitochondria. The hatching is also accompanied by the decrease of total and mitochondrial contents of oxaloacetate and of oxaloacetate/malate ratio, the activity of citrate synthase and the ratio acetyl-CoA/CoA being unchanged.     

In vitro mutagenesis of Escherichia coli citrate synthase to clarify the locations of ligand binding sites

In vitro mutagenesis techniques have been used to investigate two structure-function questions relating to the allosteric citrate synthase of Escherichia coli. The first question concerns the binding site of alpha-keto-glutarate, which is a structural analogue of the substrate oxaloacetate and yet has been suggested to be an allosteric inhibitor of the enzyme. Using oligonucleotide-directed mutagenesis of the cloned E. coli citrate synthase gene, we prepared missense mutants, designated CS226H----Q and CS229H----Q, in which histidine residues at positions 226 and 229, respectively, were replaced by glutamine. In the homologous pig heart citrate synthase it is known (Wiegand, G., and Remington, S. J. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 97-117) that the equivalent of His-229 helps to bind oxaloacetate, while the equivalent of His-226 is nearby. Kinetic and ligand binding measurements showed that CS226H----Q had a reduced affinity for oxaloacetate and alpha-ketoglutarate, while CS229H----Q bound oxaloacetate even less effectively, and was not inhibited by alpha-ketoglutarate at all under our conditions. This parallel loss of binding affinities for oxaloacetate and alpha-ketoglutarate, in two mutants altered in residues at the active site of E. coli citrate synthase, strongly suggests that inhibition of this enzyme by alpha-ketoglutarate is not allosteric but occurs by competitive inhibition at the active site. The second question investigated was whether the known inhibition by acetyl-CoA of binding of NADH, an allosteric inhibitor of E. coli citrate synthase, occurs heterotropically, as an indirect result of acetyl-CoA binding at the active site, or directly, by competition at the allosteric NADH binding site. Using existing restriction sites in the cloned E. coli citrate synthase gene, we prepared a deletion mutant which lacked 24 amino acids near what is predicted to the acetyl-CoA-binding portion of the active site. The mutant protein was inactive, and acetyl-CoA did not bind to the active site but still inhibited NADH binding. Thus acetyl-CoA can interact with both the allosteric and the active sites of this enzyme.     

Molecular biology of cytosolic acetyl-CoA generation

ATP citrate lyase (ACL) catalyses the ATP-dependent reaction between citrate and CoA to form oxaloacetate and acetyl-CoA. Our molecular characterizations of the cDNAs and genes coding for the Arabidopsis ACL indicate that the plant enzyme is heteromeric, consisting of two dissimilar subunits. The A subunit is homologous to the N-terminal third of the animal ACL, and the B subunit is homologous to C-terminal two-thirds of the animal ACL. Using both ACL-A- and ACL-B-specific antibodies and activity assays we have shown that ACL is located in the cytosol, and is not detectable in the plastids, mitochondria or peroxisomes. During seed development, ACL-A and ACL-B mRNA accumulation is co-ordinated with the accumulation of the cytosolic homomeric acetyl-CoA carboxylase mRNA. Antisense Arabidopsis plants reduced in ATP citrate lyase activity show a complex phenotype, with miniaturized organs, small cell size, aberrant plastid morphology and reduced cuticular wax. Our results indicate that ACL generates the cytosolic pool of acetyl-CoA, which is the substrate required for the biosynthesis of a variety of phytochemicals, including cuticular waxes and flavonoids.     

Elimination of CoASH interference from acetyl-CoA cycling assay by maleic anhydride

A method for the removal of CoASH from tissue extracts by maleic anhydride is described. It eliminates CoASH interference in the acetyl-CoA cycling assay using phosphotransacetylase and citrate synthase. Maleyl-CoA thioether does not hydrolyze under the conditions of the assay and allows a reduction in the number of blank samples during acetyl-CoA determination. The levels of acetyl-CoA in whole rat brain, isolated synaptosomes, and mitochondria were found to be 61, 8.6, and 31.3 pmol/mg of protein, respectively.     

Hysteretic behaviour of citrate synthase. The reaction mechanism and the exclusion of synthase being a hysteretic enzyme

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.     

Hepatic zonation of acetyl-CoA carboxylase activity.

The activities of several hepatic enzymes are preferentially zonated to the periportal or perivenous cells of the liver acinus. Employing dual-digitonin-pulse perfusion of rat liver in the study of acetyl-CoA carboxylase (ACC), we have identified a heretofore unrecognized feature of hepatic zonation, namely an intrahepatic gradient in enzyme specific activity. ACC activity shows a relative periportal localization in normally feeding rats, even when corrected for ACC protein mass. In contrast with results previously reported by us [Evans, Quistorff & Witters (1989) Biochem. J. 259, 821-829], the total mass of both hepatic ACC isoenzymes was not found to differ between the two hepatic zones in the present study. In perfusion eluates from fed animals, periportal ACC displays enhanced citrate reactivity and two kinetic components of acetyl-CoA reactivity; the largest periportal/perivenous gradient (5-fold) is accounted for by a species with a lower Km for acetyl-CoA. The zonal gradient in ACC maximal velocity, measured in eluates from fed rats, does not persist after ACC purification, although the isolated periportal enzyme, like dephosphorylated ACC, has a lower activation constant for citrate. Total ACC protein phosphatase activity is higher in periportal eluates, but no differences in the activities of either a 5'-AMP-activated ACC kinase or the cyclic-AMP-dependent protein kinase are noted between the hepatic zones. The induction of total hepatic ACC mass and specific activity, on fasting/refeeding with a high-carbohydrate diet, abolishes the periportal/perivenous activity gradient, largely owing to a selective activation of perivenous enzyme. Nutritional induction is also accompanied by a marked alteration in ACC acetyl-CoA kinetics and abolition of the gradient in total ACC phosphatase. These studies indicate that hepatic enzyme zonation, which is often attributed to differential expression of enzyme protein, may result from zonal variations in enzyme specific activity, owing to differences in allosteric regulation and/or covalent modification.     

The NADP-dependent malic enzyme MaeB is a central metabolic hub controlled by the acetyl-CoA to CoASH ratio

Malic enzymes participate in key metabolic processes, the MaeB-like malic enzymes carry a catalytic inactive phosphotransacetylase domain whose function remains elusive. Here we show that acetyl-CoA directly binds and inhibits MaeB-like enzymes with a saturable profile under physiological relevant acetyl-CoA concentrations. A MaeB-like enzyme from the nitrogen-fixing bacterium Azospirillum brasilense, namely AbMaeB1, binds both acetyl-CoA and unesterified CoASH in a way that inhibition of AbMaeB1 by acetyl-CoA is relieved by increasing CoASH concentrations. Hence, AbMaeB1 senses the acetyl-CoA/CoASH ratio. We revisited E. coli MaeB regulation to determine the inhibitory constant for acetyl-CoA. Our data support that the phosphotransacetylase domain of MaeB-like enzymes senses acetyl-CoA to dictate the fate of carbon distribution at the phosphoenol-pyruvate / pyruvate / oxaloacetate metabolic node.     

Determination of pyruvate dehydrogenase and acetyl-CoA synthetase activities using citrate synthase

Methods are described for the assay of pyruvate dehydrogenase and acetyl-CoA synthetase activities in rat brain subcellular fractions. Citrate synthase and oxaloacetate serve as a trapping system in these assays. The methods permit the determination of a large number of samples of different turbidity with satisfactory precision. Highest activities of pyruvate dehydrogenase and acetyl-CoA synthetase (117.7 and 7.29 nmol/min/mg of protein, respectively) were found in rat brain mitochondria. A three times lower activity of acetyl-CoA synthetase and negligible of pyruvate dehydrogenase was found in brain cytosol.     

Engineered citrate synthase improves citramalic acid generation in Escherichia coli

The microbial product citramalic acid (citramalate) serves as a five-carbon precursor for the chemical synthesis of methacrylic acid. This biochemical is synthesized in Escherichia coli directly by the condensation of pyruvate and acetyl-CoA via the enzyme citramalate synthase. The principal competing enzyme with citramalate synthase is citrate synthase, which mediates the condensation reaction of oxaloacetate and acetyl-CoA to form citrate and begin the tricarboxylic acid cycle. A deletion in the gltA gene coding citrate synthase prevents acetyl-CoA flux into the tricarboxylic acid cycle, and thus necessitates the addition of glutamate. In this study the E. coli citrate synthase was engineered to contain point mutations intended to reduce the enzyme's affinity for acetyl-CoA, but not eliminate its activity. Cell growth, enzyme activity and citramalate production were compared in several variants in shake flasks and controlled fermenters. Citrate synthase GltA[F383M] not only facilitated cell growth without the presence of glutamate, but also improved the citramalate production by 125% compared with the control strain containing the native citrate synthase in batch fermentation. An exponential feeding strategy was employed in a fed-batch process using MEC626/pZE12-cimA harboring the GltA[F383M] variant, which generated over 60 g/L citramalate with a yield of 0.53 g citramalate/g glucose in 132 hr. These results demonstrate protein engineering can be used as an effective tool to redirect carbon flux by reducing enzyme activity and improve the microbial production of traditional commodity chemicals.     

Structural Basis for Bacterial Quorum Sensing-mediated Oxalogenesis

The Burkholderia species utilize acetyl-CoA and oxaloacetate, substrates for citrate synthase in the TCA cycle, to produce oxalic acid in response to bacterial cell to cell communication, called quorum sensing. Quorum sensing-mediated oxalogenesis via a sequential reaction by ObcA and ObcB counteracts the population-collapsing alkaline pH of the stationary growth phase. Thus, the oxalic acid produced plays an essential role as an excreted public good for survival of the group. Here, we report structural and functional analyses of ObcA, revealing mechanistic features distinct from those of citrate synthase. ObcA exhibits a unique fold, in which a (β/α)₈-barrel fold is located in the C-domain with the N-domain inserted into a loop following α1 in the barrel fold. Structural analyses of the complexes with oxaloacetate and with a bisubstrate adduct indicate that each of the oxaloacetate and acetyl-CoA substrates is bound to an independent site near the metal coordination shell in the barrel fold. In catalysis, oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr³²² as a general base, which then attacks the thioester carbonyl carbon of acetyl-CoA to yield a tetrahedral adduct between the two substrates. Therefore, ObcA catalyzes its reaction by combining the enolase and acetyltransferase superfamilies, but the presence of the metal coordination shell and the absence of general acid(s) produces an unusual tetrahedral CoA adduct as a stable product. These results provide the structural basis for understanding the first step in oxalogenesis and constitute an example of the functional diversity of an enzyme for survival and adaptation in the environment.     

Both insulin and epidermal growth factor stimulate lipogenesis and acetyl-CoA carboxylase activity in isolated adipocytes. Importance of homogenization procedure in avoiding artefacts in acetyl-CoA carboxylase assay.

Epidermal growth factor (EGF) stimulates lipogenesis by 3-4-fold in isolated adipocytes, with a half-maximal effect at 10 nM-EGF. In the same batches of cells insulin stimulated lipogenesis by 15-fold. Freezing and prolonged homogenization of adipocytes results in release of large quantities of pyruvate carboxylase from broken mitochondria, and sufficient pyruvate can be carried through into assays for this enzyme to cause significant interference with assays of acetyl-CoA carboxylase in crude adipocyte extracts. This may account for the high amount of citrate-independent acetyl-CoA carboxylase activity reported to be present in adipocyte extracts in some previous publications. This problem may be eliminated by homogenizing very briefly without freezing. By using the modified homogenization procedure, EGF treatment of adipocytes was shown to produce an effect on acetyl-CoA carboxylase activity almost identical with that of insulin. Both messengers increase Vmax. without significant effect on the Ka for the allosteric activator, citrate.