Quantitation of the interaction between citrate synthase and malate dehydrogenase

Formation of a bienzyme complex of pig heart mitochondrial malate dehydrogenase and citrate synthase in a buffered system is demonstrated by means of a covalently attached fluorescent probe to citrate synthase. Assuming 1:1 stoichiometry of the enzymes in the complex, an apparent dissociation constant of 10(-6) M was calculated from fluorescence anisotropy measurements. The effect of various metabolites on the interaction was tested. NAD+, oxalacetate, citrate, ATP, and L(-)- or D(+)-malate had no effect on the association of the two enzymes, whereas alpha-ketoglutarate increased and NADH decreased it. The interaction of mitochondrial citrate synthase with cytosolic malate dehydrogenase was found to be much weaker, whereas interaction of citrate synthase with another cytosolic enzyme, aldolase, could not be detected. In kinetic experiments, the activation of malate dehydrogenase by citrate synthase was observed. The effect of pyridine nucleotides and alpha-ketoglutarate is discussed in relation to the direction of the metabolic flow of oxalacetate.     

Regulatory properties of citrate synthase from Rickettsia prowazekii

Citrate synthase [citrate (si)-synthase] (EC 4.1.3.7) was partially purified from extracts of highly purified typhus rickettsiae (Rickettsia prowazekii). Molecular exclusion and affinity column chromatography were used to prepare 200-fold-purified citrate synthase that contained no detectable malate dehydrogenase (EC 1.1.1.37) activity. Rickettsial malate dehydrogenase also was partially purified (200-fold) via this purification procedure. Catalytically active citrate synthase exhibited a relative molecular weight of approximately 62,000 after elution from a calibrated Sephacryl S-200 column. Acetyl coenzyme A saturation of partially purified enzyme was sensitive to strong competitive inhibition with adenylates (ATP greater than ADP much greater than AMP). [beta,gamma-methylene]ATP, dATP, and dADP also caused strong inhibition, but guanosine and cytosine nucleotides were significantly less inhibitory. Adenylates had no effect on oxalacetate saturation kinetics when acetyl coenzyme A was present in high concentration (greater than or equal to 50 microM). Neither NADH nor alpha-ketoglutarate affected the saturation kinetics of rickettsial citrate synthase. Thus, citrate synthase from R. prowazekii exhibits greater similarity to the eucaryotic and gram-positive procaryotic enzymes than to citrate synthase from free-living gram-negative bacteria. These results represent the first characterization of a highly purified key regulatory enzyme from these obligate intracellular parasitic bacteria.     

Effect of mitogens on the maximum activities of hexokinase, lactate dehydrogenase, citrate synthase and glutaminase in rat mesenteric lymph node lymphocytes and splenocytes during the early period of culture

1. The activities of hexokinase, lactate dehydrogenase and citrate synthase were maintained in mesenteric lymph node lymphocytes during 4 hr of culture: the activity of glutaminase increased during this period of time. 2. In splenocytes, the activity of hexokinase decreased markedly during the 4 hr period, whereas those of lactate dehydrogenase and glutaminase remained constant, and that of citrate synthase increased dramatically. 3. In both mesenteric lymphocytes and splenocytes, addition of the T-cell mitogens, phytohaemagglutinin or concanavalin-A, to the culture medium caused decreases in the activities of both hexokinase and citrate synthase. 4. In contrast, these mitogens increased the activity of glutaminase in both cell types. 5. Addition of the B-cell mitogen, bacterial lipopolysaccharide, had little effect on hexokinase, lactate dehydrogenase or citrate synthase but increased markedly that of glutaminase in mesenteric lymph node lymphocytes. 6. In splenocytes this mitogen prevented much of the decrease in hexokinase activity, increased the activities of citrate synthase and glutaminase but had little effect on that of lactate dehydrogenase.     

An investigation of the mechanism of activation of tryptophan by tryptophanyl-tRNA synthetase from beef pancreas

Exposure of dark-grown Euglena to white or red light, but not blue light, produced a twofold increase in the specific activity of citrate synthase. A 400-fold purification of mitochondrial citrate synthase (subunit Mr = 44000) was achieved from cells of Euglena gracilis by affinity chromatography on ATP-activated agarose. Antisera, raised against the homogeneously pure enzyme, were used to demonstrate that the increase in citrate synthase activity on exposure of dark-grown cells to light resulted from an increase in citrate synthase protein. Anti-(citrate synthase) was used to detect precursor citrate synthase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. Citrate synthase mRNA was found to be present in cells at all stages of regreening. However, extraction and translation of polyadenylated RNA from free polysomes isolated from darkgrown and regreening cells demonstrated that appreciable translation of citrate synthase mRNA was only occurring in regreening cells.     

Regulation of citrate synthase activity of Saccharomyces cerevisiae.

Citrate synthase activity of Saccharomyces cerevisiae was determined by a radioactive assay procedure and the reaction product, 14C-citric acid, was identified by chromatographic techniques. ATP, d-ATP, GTP and NADH were most inhibitory to the citrate synthase invitro. The activity was inhibited to a lesser extent by ADP, UTP, and NADP whereas, AMP and CTP were much less inhibitory. NADH, like NAD, glutamic acid, glutamine, arginine, ornithine, proline, aspartic acid and alpha-ketoglutarate exhibited no inhibition. These results have been discussed in the light of the role of citrate synthase for the energy metabolism and glutamic acid biosynthesis.     

Effects of altered citrate synthase and isocitrate dehydrogenase expression on internal citrate concentrations and citrate efflux from tobacco (Nicotiana tabacum L.) roots

To assess the effectiveness of manipulating citrate metabolism with the aim of increasing citrate efflux from roots, we generated transgenic tobacco (Nicotiana tabacum L.) lines that either overexpressed mitochondrial citrate synthase (EC 4.1.3.7) activity or had reduced activity of cytosolic isocitrate dehydrogenase (EC 1.1.1.42). Despite increases in citrate synthase activities in transgenic lines of up to 5-fold, neither internal citrate concentrations nor citrate efflux were increased compared to controls suggesting that, in tobacco, citrate synthase activity does not directly determine citrate accumulation and efflux. Consistent with a lack of effect on citrate efflux, the increase in citrate synthase activity did not enhance the aluminium resistance of the transgenic lines. Preliminary data collected on two transgenic lines with cytosolic isocitrate dehydrogenase activities reduced to one-tenth and one third of the control for shoot and root tissues respectively, showed that while these changes in activities were associated with a 1.5-fold increase in internal citrate concentrations of both types of tissue, citrate efflux from roots was not increased. Further work is needed to establish whether the increase in internal citrate concentration is associated with enhanced aluminium resistance of these lines. We conclude that in tobacco internal citrate concentrations and citrate efflux are largely insensitive to large changes in either mitochondrial citrate synthase or cytosolic isocitrate dehydrogenase activities and suggest that other factors, such as transport out of the roots, control citrate efflux.     

Interactions of citrate synthases from osmoconforming and osmoregulating animals with salt: possible signs of molecular eco-adaptation?

This study considers differential sensitivity of citrate synthase (citrate oxaloacetatelyase [CoA acetylating]) EC 4.1.3.7. from an osmoconforming animal (sea anemone) and an osmoregulating animal (the pig) to salt. Attention is drawn to the fact that the osmoconforming sea anemone is in essence a sessile creature while the pig is readily mobile and able to change its ionic environment at will. It had been shown earlier that citrate synthase from another osmoconformer (oyster) is also not sensitive to ionic strength while citrate synthase from osmoregulating white shrimp is sensitive to increasing levels of salt. However, these enzymes are characteristically regulated by ATP and alpha-ketoglutarate. Both forms of citrate synthase are denatured by 6 M guanidine hydrochloride and are aided by salt levels in their refolding but the rate and extent of refolding of the osmoconformer citrate synthase are greater than those of the osmoregulator citrate synthase. Catalytic activity of both forms of citrate synthase is inhibited by incubation in distilled water; osmoconformer citrate synthase was inhibited completely in 7 h while osmoregulator citrate synthase was inhibited only 60% in this time and 80% after 22 h in distilled water. The eco-adaptive and evolutionary implications of these findings are discussed.     

Subunit equilibria of porcine heart citrate synthase. Effects of enzyme concentration, pH, and substrates

Porcine heart citrate synthase, a dimeric protein of Mr = 100,000 composed of two identical subunits, is shown to undergo a monomer-dimer equilibrium. The extent of dimerization is found to be dependent on the concentration of citrate synthase, pH, ionic strength, and the specific buffer system employed. Oxaloacetate and citrate, substrates for the forward and reverse reaction catalyzed by citrate synthase, affect dimerization at concentrations of the protein which exists as monomer in their absence. The dissociation of citrate synthase dimers has been demonstrated utilizing the techniques of gel permeation chromatography, fluorescence polarization, fluorescence energy transfer, and heat denaturation. Earlier studies of citrate synthase quarternary structure found the protein to be nondissociable except under denaturing conditions or extensive modification; however, most former studies were performed at relatively high protein concentration, ionic strength, and pH, conditions which stabilize the dimer. In light of recent evidence derived from x-ray crystallographic studies showing amino acid residues from one subunit contributing to the citrate and CoA binding sites of the other, the dissociation into monomers would be expected to have profound effects on citrate synthase activity and regulation, as well as overall tricarboxylic acid cycle activity.     

A comparison of the citrate synthases of Escherichia coli and Acinetobacter anitratum

Citrate synthase has been purified to homogeneity from a strain of the Gram-negative aerobic bacterium Acinetobacter anitratum in a form which retains its sensitivity to the allosteric inhibitor NADH. In subunit size, amino acid composition, and antigenic reactivity the enzyme shows a marked structural resemblance to the citrate synthase of the Gram-negative facultative anaerobe Escherichia coli. Whereas the E. coli enzyme is subject to a strong, hyperbolic inhibition by NADH (Hill's number n = 1.0, Ki = 2 microM), the A. anitratum enzyme shows a weak, sigmoid response (n = 1.6, I0.5 = 140 microM) to this nucleotide. With E. coli, NADH inhibition is competitive with acetyl-CoA, and noncompetitive with oxaloacetate; with A. anitratum, NADH is noncompetitive with both substrates. Acinetobacter anitratum citrate synthase shows hyperbolic saturation with acetyl-CoA (n = 1.8). The finding of Weitzman and Jones (Nature (London) 219, 270 (1968) that NADH inhibition of the enzyme from Acinetobacter spp. is reversible by AMP, while that from E. coli is not, is explained by the much greater affinity of the E. coli enzyme for NADH. Unlike E. coli citrate synthase, the A. anitratum enzyme does not react with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of denaturation. With a second sulfhydryl reagent, 4,4'-dithiodipyridine (4,4'-PDS), the A. anitratum enzyme reacts with 1 equiv. of subunit; this modification induces a partial activity loss (attributable to a arise in the Km for acetyl-CoA) and an increase in the sensitivity to NADH. With the E. coli enzyme, 4,4'-PDS causes complete inactivation. Acinetobacter anitratum citrate synthase is much more resistant to urea denaturation than the E. coli enzyme is; the resistance of both enzymes to urea is greatly improved in the presence of 1 M KCl. It is suggested that the amino acid sequences of the subunits of the citrate synthases of these two bacteria are about 90% homologous, and that the 10% differences are in key residues, perhaps largely in the subunit contact regions, which account for the differences in allosteric properties.     

Fatty acid oxidation in human and rat heart. Comparison of cell-free and cellular systems

Oxidation rates of palmitate and activities of the mitochondrial marker enzymes cytochrome c oxidase and citrate synthase have been determined in homogenates, isolated mitochondria and slices of human and rat heart and in calcium-tolerant rat cardiac myocytes. Homogenates and mitochondria from rat heart showed a 6- and 2.5-fold higher palmitate oxidation rate than the corresponding preparations from human heart. From the palmitate oxidation rates and cytochrome c oxidase and citrate synthase activities as parameters, the mitochondrial protein contents of human and rat heart were calculated to be about 18 and 45 mg/g wet weight, respectively. Based on citrate synthase activities, the fatty acid oxidation rates were about the same in homogenates and isolated mitochondria, much lower in myocytes and lowest in slices. In the cellular systems the palmitate molecule was more completely oxidized than in homogenates or isolated mitochondria. Fatty acid oxidation rates were concentration-dependent in slices, but not with myocytes. With the cellular systems, palmitate oxidation was synergistically stimulated by the addition of carnitine, coenzyme A and ATP to the incubation medium. This stimulation could be attributed only partly to an increased oxidation in damaged cells.     

Characterization of citrate synthase from Geobacter sulfurreducens and evidence for a family of citrate synthases similar to those of eukaryotes throughout the Geobacteraceae

Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (kcat= 8.3 s(-1); Km= 14.1 and 4.3 microM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (Ki= 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K+ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.     

Site-directed mutagenesis of citrate synthase; the role of the active-site aspartate in the binding of acetyl-CoA but not oxaloacetate

Asp-362, a potential key catalytic residue of Escherichia coli citrate synthase (citrate oxaloacetate-lyase [pro-3S)-CH2COO- ----acetyl-CoA), EC 4.1.3.7) has been converted to Gly-362 by oligonucleotide-directed mutagenesis. The mutant gene was completely sequenced, using a series of synthetic oligodeoxynucleotides spanning the structural gene to confirm that no additional mutations had occurred during genetic manipulation. The mutant gene was expressed in M13 bacteriophage and produced a protein which migrated in an identical manner to wild-type E. coli citrate synthase on SDS-polyacrylamide gels and which cross-reacted with E. coli citrate synthase antiserum. The mutant gene was subsequently recloned into pBR322 for large scale purification of the protein, and the resulting plasmid, pCS31, used to transform the citrate synthase deletion strain, W620. The mutant enzyme purified in an analogous manner to wild-type E. coli citrate synthase and expressed less than 2% of wild-type enzyme activity. The activity of the partial reactions catalysed by citrate synthase was similarly affected suggesting that this residual activity may be due to contaminating wild-type enzyme activity. The mutant citrate synthase retains a high-affinity NADH-binding site consistent with the protein preserving its overall structural integrity. Oxaloacetate binding to the protein is unaffected by the Asp-362 to Gly-362 mutation. Binding of the acetyl-CoA analogue, carboxymethyl-CoA, could not be detected in the mutant protein indicating that the lack of catalytic competence is due primarily to the inability of the protein to bind the second substrate, acetyl-CoA.     

Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal.

The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.     

Disruption of the peroxisomal citrate synthase CshA affects cell growth and multicellular development in Dictyostelium discoideum

Non-mitochondrial citrate synthase catalyses citrate synthesis in the glyoxylate cycle in gluconeogenesis. Screening Dictyostelium discoideum mutants generated by insertional mutagenesis isolated a poor-growing mutant that displayed aberrant developmental morphology on bacterial lawns. Axenically grown mutants developed normally and formed mature fruiting bodies on buffered agar. The affected locus encoded a novel protein (CshA) that was homologous to glyoxysomal citrate synthase. cshA was expressed maximally during vegetative growth and gradually decreased through subsequent developmental stages. An in vitro citrate synthase assay revealed that cshA disruption resulted in a 50% reduction in enzyme activity, implicating CshA as an active citrate synthase. The amino-terminus of CshA was found to have an atypical mitochondrial targeting signal, instead containing a unique nonapeptide sequence (RINILANHL) that was homologous to the conserved peroxisomal targeting signal 2 (PTS2). CshA protein was shown to be localized in the peroxisomes, and the RINILANHL sequence only efficiently targeted the peroxisomal green fluorescent protein. The growth defect of cshA(-) cells was associated with the impairment of phagocytosis and fluid-phase endocytosis, independent from cytokinesis. Disrupted multicellular development on bacterial lawns resulted from the abnormal susceptibility to the environmental conditions, perhaps because of citrate insufficiency. Taken together, these results provide new insights into the function of peroxisomal citrate synthase in cell growth and multicellular development.     

Action of aldosterone on citrate synthase in cultured renal collecting duct cells

Cultured renal collecting duct cells from neonatal rabbit kidney were used to examine the influence of aldosterone on enzymatic activity of citrate synthase during increase in Na+ transport. Control epithelia showed citrate synthase activity of 71 +/- 3 mU/mg protein (n = 28), while after aldosterone treatment citrate synthase activity was significantly increased to 79 +/- 6 mU/mg at 1 h (n = 5), to 88 +/- 6 mU/mg at 2 h (n = 6) and to 93 +/- 8 mU/mg protein at 3 h (n = 5). Citrate synthase activity subsequently decreased to basal values. Spironolactone fully blocked the aldosterone-induced increase in citrate synthase activity. The time course of enzyme stimulation after aldosterone administration indicates that the hormone activates citrate synthase during the physiological early response phase.     

The CIT3 gene of Saccharomyces cerevisiae encodes a second mitochondrial isoform of citrate synthase

We have identified a third citrate synthase gene in Saccharomyces cerevisiae which we have called CIT3 Complementation of a citrate synthase-deficient strain of Escherichia coli by lacZ  :: CIT3 gene fusions demonstrated that the CIT3 gene encodes an active citrate synthase. The CIT3 gene seems to be regulated in the same way as CIT1 , which encodes the mitochondrial isoform of citrate synthase. Deletion of the CIT3 gene in a Δ cit1 background severely reduced growth on the respiratory substrate glycerol, whilst multiple copies of the CIT3 gene in a Δ cit1 background significantly improved growth on acetate. In vitro import experiments showed that cit3p is transported into the mitochondria. Taken together, these data show that the CIT3 gene encodes a second mitochondrial isoform of citrate synthase.     

Organelle-specific isozymes of citrate synthase in the endosperm of developing ricinus seedlings

Chromatographic analysis of organelle-associated citrate synthase activity revealed distinct mitochondrial and glyoxysomal forms of the enzyme. The chromatographic elution patterns on hydroxylapatite, carboxymethylcellulose and DEAE-cellulose of citrate synthase from the endosperm of 4.5-day-old castor bean seedlings revealed significant differences for mitochondrial and glyoxysomal activities of the enzyme. The endoplasmic reticulum-associated citrate synthase activity eluted from DEAE-cellulose in a pattern that was identical to that of the glyoxysomal activity. The same kinds of organelle specific isozyme elution patterns were observed with young, developing seedlings. Gibberellic acid-treatment of young seedlings increased total recoverable citrate synthase activity from endosperm tissue but did not modify the organelle specific isozyme relationships.     

Rhodothermus marinus: a thermophilic bacterium producing dimeric and hexameric citrate synthase isoenzymes

Two separate citrate synthases from the extremely thermophilic bacterium Rhodothermus marinus have been identified and purified. One of the enzymes is a hexameric protein and is the first thermostable, hexameric citrate synthase to be isolated. The other is a dimeric enzyme, which is also thermostable but possesses both citrate synthase and 2-methyl citrate synthase activities. 2-Methyl citrate synthase uses propionyl-coenzyme A as one of its substrates and in Escherichia coli, for example, it has been implicated in the metabolism of propionate. However, no growth of R. marinus was observed using minimal medium with propionate as the sole carbon source, and both hexameric and dimeric enzymes were produced irrespective of whether propionate was included in the growth medium. The data are discussed with respect to the evolutionary relationships between the known hexameric and dimeric citrate synthases and 2-methyl citrate synthase.     

Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-CoA hydrolase.

1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl-CoA + oxaloacetate + H2O----citrate + CoASH) to 4% but did not affect the hydrolysis of citryl-CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well. 2. Tryptic hydrolysis occurs at the N-terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues. 3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue-Sepharose and differentiated by their isoelectric points (pI = 6.68 +/- 0.08 and pI = 6.37 +/- 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N-terminus which is blocked in the native enzyme only. 4. Edman degradation of the large tryptic fragment yielded the N-terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N-terminal region (positions 9-32) of citrate synthase from Thermoplasma acidophilum. 5. The conversion of citrate synthase into essentially a citryl-CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N-terminal small peptide.     

Complexes between mitochondrial enzymes and either citrate synthase or glutamate dehydrogenase

Experiments performed in polyethylene glycol and with a divalent crosslinker indicate that both mitochondrial malate dehydrogenase and aspartate aminotransferase can form hetero enzyme—enzyme complexes with either glutamate dehydrogenase or citrate synthase. In general, these as previous results indicate that complexes with the aminotransferase are favored over those with malate dehydrogenase and complexes with glutamate dehydrogenase are favored over those with citrate synthase. When the levels of enzymes are low, the only detectable complex is between the aminotransferase and glutamate dehydrogenase. Under these conditions, palmitoyl-CoA is required for complexes between the other three enzyme pairs, however, palmitoyl-CoA also enhances interactions between glutamate dehydrogenase and the aminotransferase. DPNH disrupts complexes with malate dehydrogenase and has little effect on those with the aminotransferase, while oxalacetate disrupts complexes with citrate synthase but has little effect on those with glutamate dehydrogenase. The citrate synthase-aminotransferase complex was favored in the presence of DPNH plus malate, which disrupt the other three enzyme-enzyme complexes. Glutamate dehydrogenase has a higher affinity and capacity than citrate synthase for palmitoyl-CoA. Consequently, lower levels of palmitoyl-CoA are required to enhance interactions with glutamate dehydrogenase. Furthermore, glutamate dehydrogenase can compete with citrate synthase for palmitoyl-CoA and thus can prevent palmitoyl-CoA from enhancing interactions between citrate synthase and either malate dehydrogenase or the aminotransferase.