Phosphorylation of ATP citrate lyase in response to glucagon.

Incubation of hepatocytes with [32P]orthophosphate resulted in the incorporation of 32P into material that is precipitated by reaction with antibodies to ATP citrate lyase. The amount of radioactivity precipitated was decreased when unlabeled, purified ATP citrate lyase was added to extracts of hepatocytes that had been incubated with [32P]orthophosphate. Addition of glucagon to hepatocytes that had been preincubated with [32P]orthophosphate resulted in a 56% increase in acid-stable 32P in the trichloroacetic acid-insoluble portion of immunoprecipitates. Catalytic phosphate bound to ATP citrate lyase reaction with ATP and Mg2+ is acid-labile; thus, glucagon-dependent phosphorylation is distinguished from the catalytic phosphate. When hepatocytes were incubated in the absence of [32P]orthophosphate and extracted in a medium containing [gamma-32P]ATP, no acid-stable 32P was present in immunoprecipitates. This indicates that the incorporation into ATP citrate lyase of acid-stable phosphate occurs prior to extraction of the enzyme. Preliminary studies, using a procedure that allows for measurement of enzyme activity starting 1 min after beginning the extraction of lyase from hepatocytes, have shown no difference in lyase activity when hepatocytes are treated with or without glucagon.     

ATP:citrate lyase of Rhodotorula gracilis: purification and properties

ATP:citrate lyase was purified from the oleaginous yeast Rhodotorula gracilis to homogeneity as judged by polyacrylamide gel electrophoresis, using a novel citrate-Sepharose procedure. The enzyme was found to have a molecular weight of 520,000 and consisted of four identical subunits (Mr = 120,000). Two minor low molecular weight bands were observed on SDS-PAGE (Mr 51,000 and 49,000). Trypsin digestion experiments indicated that these could have been the result of limited proteolysis by an endogenous trypsin-like proteinase. In this respect, it resembles the mammalian ATP:citrate lyase. The enzyme was stimulated by NH+4 ions and inhibited by palmitoyl, lauroyl, oleoyl, myristoyl and stearoyl-CoA esters, glutamate and glucose 6-phosphate but not by acetyl-CoA or shorter chain fatty acyl-CoA esters. The enzyme exhibited normal Michaelis-Menten kinetics for citrate; however there was a 3-fold increase in Km with a high concentration of Cl- ions (0.25 M). The possible regulatory roles of ATP:citrate lyase in R. gracilis are discussed in the light of these findings.     

Evidence for an essential arginine residue at the active site of ATP citrate lyase from rat liver.

Rat liver ATP citrate lyase was inactivated by 2, 3-butanedione and phenylglyoxal. Phenylglyoxal caused the most rapid and complete inactivation of enzyme activity in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid buffer, pH 8. Inactivation by both butanedione and phenylglyoxal was concentration-dependent and followed pseudo- first-order kinetics. Phenylglyoxal also decreased autophosphorylation (catalytic phosphate) of ATP citrate lyase. Inactivation by phenylglyoxal and butanedione was due to the modification of enzyme arginine residues: the modified enzyme failed to bind to CoA-agarose. The V declined as a function of inactivation, but the Km values were unaltered. The substrates, CoASH and CoASH plus citrate, protected the enzyme significantly against inactivation, but ATP provided little protection. Inactivation with excess reagent modified about eight arginine residues per monomer of enzyme. Citrate, CoASH and ATP protected two to three arginine residues from modification by phenylglyoxal. Analysis of the data by statistical methods suggested that the inactivation was due to modification of one essential arginine residue per monomer of lyase, which was modified 1.5 times more rapidly than were the other arginine residues. Our results suggest that this essential arginine residue is at the CoASH binding site.     

Radicicol binds and inhibits mammalian ATP citrate lyase

Six different biotinylated radicicol derivatives were synthesized as affinity probes for identification of cellular radicicol-binding proteins. Derivatives biotinylated at the C-17 (BR-1) and C-11 (BR-6) positions retained the activity of morphological reversion in v-src-transformed 3Y1 fibroblasts. Two radicicol-binding proteins, 120 and 90-kDa in size, were detected in HeLa cell extracts by employing BR-1 and BR-6, respectively. The 90-kDa protein bound to BR-6 was identified to be Hsp90 by immunoblotting. The 120-kDa protein bound to BR-1 was purified from rabbit reticulocyte lysate, and its internal amino acid sequence was identical to that of human and rat ATP citrate lyase. The identity of the 120-kDa protein as ATP citrate lyase was confirmed by immunoblotting. Interaction between BR-1 and ATP citrate lyase was blocked by radicicol but not by herbimycin A that interacts with Hsp90. These results suggest that radicicol binds the two proteins through different molecular portions of its structure. BR-1-bound ATP citrate lyase isolated from rabbit reticulocyte lysate showed no enzymatic activity. The activity of rat liver ATP citrate lyase was inhibited by radicicol and BR-1 but not by BR-6. Kinetic analysis demonstrated that radicicol was a non-competitive inhibitor of ATP citrate lyase with K(i) values for citrate and ATP of 13 and 7 microm, respectively.     

Association of ATP citrate lyase with mitochondria

(1) The association of ATP citrate lyase with mitochondria was studied with isolated rat hepatocytes and mitochondria. (2) When hepatocytes were treated with digitonin, about 25% of the lyase activity was released like a mitochondrial enzyme. (3) The effect of temperature on release of lyase from hepatocytes was different from that on the release of other cytosolic or mitochondrial enzymes. (4) The fraction of total hepatic lyase in mitochondrial preparations made with exogenous MgCl₂ was 30 times greater than that for a cytosolic marker enzyme, phosphoglycerate kinase. (5) Lyase substrates enhanced the release of the enzyme both from hepatocytes and from isolated mitochondria. (6) The metabolic significance of association of ATP citrate lyase with mitochondria is discussed. (7) Data obtained in the course of these experiments indicate that less than 3% of adenylate kinase is cytosolic.     

Studies on a rat-liver cell-sap protein yielding 3-[32P]-phosphohistidine after incubation with [32P]ATP and alkaline hydrolysis. Identification of the protein as ATP citrate lyase

1. The rat-liver cell-sap material from which 3-[32P]phosphohistidine was previously isolated after incubation with [gamma-32P]ATP and alkaline hydrolysis, was shown to increase about 6-fold on a high-carbohydrate diet. This increase in 32P labelling corresponded to the increase in ATP citrate lyase activity of livers of rats fed on a high-carbohydrate diet, as reported by others. 2. ATP citrate lyase [ATP:citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephopshorylating), EC 4.1.3.8] was purified from rat liver essentially according to the method of Plowman and Cleland (J. Biol. Chem., 242 (1967) 4239). The purified enzyme was incubated for a short time at 0 degree with [gamma-32P]ATP in the presence of 20 mM magnesium acetate. The phosphorylated protein was hydrolysed in alkali and the main part of the radioactivity was identified as 3-[32P]phosphohistidine. The identity of the phosphorylated amino acid was established by Dowex-1 chromatography, paper electrophoresis, paper chromatography and by analysis of the stability to acid. 3. It is concluded from these and previous results from this laboratory that ATP citrate lyase and nucleoside diphosphate kinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) account for most of the normal rat-liver cell-sap protein which is rapidly phosphorylated by ATP.     

Stoichiometry of phosphorylation of hepatic ATP-citrate lyase by protein kinase

Hepatic ATP-citrate lyase prepared with a fluoride-free step to allow endogenous phosphatases to dephosphorylate the enzyme was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase and [γ-³²P]ATP. After electrophoresis the radioactive phosphate was located predominantly in the gel slice containing the Coomassie blue stained protein corresponding to ATP-citrate lyase. The Stoichiometry of phosphorylation of hepatic ATP-citrate lyase in vitro by the catalytic subunit was such that 0.53 ± 0.02 molecules of phosphate were incorporated per subunit. The degree of phosphorylation was independent of the amount of ATP-citrate lyase present as substrate in the concentration range 1.2–6.4 μm. In the absence of catalytic subunit there was very little labeled phosphate incorporated into ATP-citrate lyase. Phosphorylation of ATP-citrate lyase by catalytic subunit was abolished by the specific protein inhibitor of cyclic AMP-dependent protein kinase. When ATP-citrate lyase was subjected to electrophoresis under nondenaturing conditions, lyase activity was recovered from the gel slice corresponding to the Coomassie blue staining phosphoprotein of a stained gel run in parallel.     

Effects of phosphorylation on the kinetic properties of rat liver ATP-citrate lyase

Homogeneous rat liver ATP-citrate lyase (EC 4.1.3.8) was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. In agreement with other workers, the maximum level of phosphorylation that we observed was approx. 2 mol/mol of tetramer. Phosphorylated and non-phosphorylated forms of ATP-citrate lyase were prepared. Their kinetic properties were examined using an assay system in which the concentrations of Mg.ATP, magnesium.citrate and CoA were varied systematically at a constant concentration of Mg2+. The phosphorylated form had a two-fold higher Km for Mg.ATP than did the non-phosphorylated form, but no other kinetic differences between the two forms were detected. When ATP-citrate lyase was assayed at a concentration of Mg.ATP well below Km, it was found that phosphorylation of the enzyme correlated well with a decrease of approx. 50% in its activity. This is the first demonstration that phosphorylation can affect the activity of ATP-citrate lyase.     

Dependence of ATP citrate lyase activity on cell density of isolated rat hepatocytes

The activities of two enzymes involved in the lipogenic process, ATP citrate lyase and NADP-linked malic enzyme were evaluated as a function of cell density in isolated rat hepatocytes. The activity of ATP citrate lyase was profoundly affected by cell density with the activity/cell being higher at low cell densities than at high cell densities. Malic enzyme was not similarly affected, nor was cellular ATP content. The effect was observed regardless of dietary state but was most dramatic with hepatocytes from fasted-refed rats. Both an activator and an inhibitor of ATP citrate lyase have been isolated from conditioned medium from cells at low density and at high density, respectively. The activator fraction was heat stable while the inhibitor fraction was heat labile, and both factors had molecular weights in excess of 10,000 daltons.     

Calcitonin and hepatic fatty acid synthesis: effect of thyroparathyroidectomy on the elevation of ATP citrate lyase activity by refeeding of starved rats

The effect of thyroparathyroidectomy (TPTX) on ATP citrate lyase regulation, a rate-limiting enzyme of fatty acid synthesis in hepatic cytosol, was investigated in rats refed after a 24 h fast. ATP citrate lyase activity in the hepatic cytosol was increased 2-fold by refeeding. This increase was suppressed about 50% by TPTX. The suppression of the enzyme activity by TPTX was completely restored by administration of calcitonin (CT; 80 MRC mU/100 g body weight). This hormonal effect was also observed at 20 MRC mU/100 g dose of CT. CT administration to refeeding-TPTX rats produced a significant increase in the calcium content of the liver tissue and the cytosol. The cytosolic ATP citrate lyase activity increase with CT administration was completely blocked by treatment of cytosol with EGTA (10 microM). This inhibition was clearly reversed by addition of calcium ion (1.25-5.0 microM). In addition, CT-induced rise in enzyme activity was markedly reduced by the presence of W-7 (5 and 50 microM), a calmodulin inhibitor, in the enzyme assay system. The present results suggest that CT plays a role in the elevation of hepatic ATP citrate lyase activity brought about by refeeding of fasted rats, and that this hormonal regulation might depend on Ca2+-calmodulin.     

Reactivity and inhibitor potential of hydroxycitrate isomers with citrate synthase, citrate lyase, and ATP citrate lyase.

The four isomers of hydroxycitrate have been tested as substrates and inhibitors for citrate synthase, citrate lyase, and ATP citrate lyase. None of the isomers served as a substrate for citrate synthase and they were moderate to weak inhibitors of this reaction. Of the four isomers, only (pncit)-(2S)-2-hydroxycitrate did not serve as a substrate for citrate lyase while (pncit)-(4S)-4-hydroxycitrate was the only isomer which did not serve as a substrate for ATP citrate lyase. No consistent pattern of reactivity or inhibitor potency was seen with the different isomeric hydroxycitrates. It is proposed that more than one mode of binding is possible between the isomers and the three different active sites.     

Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyase.

The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl-ACL), an alpha(4)beta(4) heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent K(m) value of 0.21 +/- 0.04 mm. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (n(H)) of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 +/- 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a K(i) value of 0.037 +/- 0.006 mm. Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl-ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl-ACL. His273 of the alpha subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme. However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA.     

Multiple chromatographic forms of ATP citrate lyase from rat liver.

ATP citrate lyase is shown to exist as multiple forms in extracts of rat liver. DEAE-Sephadex ion-exchange chromatography of liver supernatants reveals two peaks of activity. A minor, basic, component, comprising 14% of the recovered activity, is eluted without retention, whereas the major, acidic, form is eluted by a KCl gradient. Gel filtration of similar extracts shows the presence of a high-Mr form of ATP citrate lyase (Mr around 10(7) in addition to the tetrameric enzyme (Mr 4.1 X 10(5). This associated state, which represents 10% of the total activity, is unstable, breaking down to the tetramer, and appears to be disrupted by Mg2+. The basic form changes in the partially purified state to give the acidic form. Most of the high-Mr enzyme is acidic in nature. No evidence could be found for an association of the enzyme with mitochondrial or microsomal membranes. ATP citrate lyase from rat brain also shows two peaks of activity on DEAE-Sephadex ion-exchange chromatography, but the activity is distributed between the peaks in almost equal proportions. However, only the tetrameric enzyme was observed on gel filtration.     

On the catalytic mechanism of human ATP citrate lyase

ATP citrate lyase (ACL) catalyzes an ATP-dependent biosynthetic reaction which produces acetyl-coenzyme A and oxaloacetate from citrate and coenzyme A (CoA). Studies were performed with recombinant human ACL to ascertain the nature of the catalytic phosphorylation that initiates the ACL reaction and the identity of the active site residues involved. Inactivation of ACL by treatment with diethylpyrocarbonate suggested the catalytic role of an active site histidine (i.e., His760), which was proposed to form a phosphohistidine species during catalysis. The pH-dependence of the pre-steady-state phosphorylation of ACL with [γ-(33)P]-ATP revealed an ionizable group with a pK(a) value of ~7.5, which must be unprotonated for the catalytic phosphorylation of ACL to occur. Mutagenesis of His760 to an alanine results in inactivation of the biosynthetic reaction of ACL, in good agreement with the involvement of a catalytic histidine. The nature of the formation of the phospho-ACL was further investigated by positional isotope exchange using [γ-(18)O(4)]-ATP. The β,γ-bridge to nonbridge positional isotope exchange rate of [γ-(18)O(4)]-ATP achieved its maximal rate of 14 s(-1) in the absence of citrate and CoA. This rate decreased to 5 s(-1) when citrate was added, and was found to be 10 s(-1) when both citrate and CoA were present. The rapid positional isotope exchange rates indicated the presence of one or more catalytically relevant, highly reversible phosphorylated intermediates. Steady-state measurements in the absence of citrate and CoA showed that MgADP was produced by both wild type and H760A forms of ACL, with rates at three magnitudes lower than that of k(cat) for the full biosynthetic reaction. The ATPase activity of ACL, along with the small yet significant positional isotope exchange rate observed in H760A mutant ACL (~150 fold less than wild type), collectively suggested the presence of a second, albeit unproductive, phosphoryl transfer in ACL. Mathematical analysis and computational simulation suggested that the desorption of MgADP at a rate of ~7 s(-1) was the rate-limiting step in the biosynthesis of AcCoA and oxaloacetate.     

Compartmentation of enzymes: ATP citrate lyase in hepatocytes from fed or fasted rats

Compared with traditional techniques of tissue homogenization, digitonin fractionation of isolated hepatocytes provides a much more rapid and, in some instances, more accurate determination of enzyme compartmentation. Results with ATP citrate lyase (EC 4.1.3.8) illustrate the information that uniquely can be obtained. Although the enzyme was previously thought to be entirely cytosolic, digitonin fractionation has shown that a portion of total cellular ATP citrate lyase is bound to mitochondria or some other structure, and the amount bound varies with the animal's nutritional state. In hepatocytes from rats that were starved for 2 days, fed NIH stock diet ab libitum, or starved for 2 days and then refed a fat-free diet for 2 days, the noncytosolic activity was, respectively, 52, 21, or 24% of total cellular lyase. However, because starvation/refeeding greatly induces lipogenic enzymes, the amount of bound lyase activity in this dietary state was 10-12 times greater than that in rats that were starved or fed ad libitum. The association of citrate lyase with a subcellular organelle is also influenced by CoA. Addition of 20 microM CoA to the digitonin fractionation medium caused all of the lyase to be released from cells like a cytosolic enzyme. Conversely, when cellular free CoA was decreased by incubating hepatocytes with the hypolipidemic agent 5-(tetradecyloxy)-2-furoic acid, the amount of bound lyase was increased. These results suggest the possibility that the noncytosolic ATP citrate lyase may have a special role in lipogenesis.     

Purification and characterization of ATP:citrate lyase from Hydrogenobacter thermophilus TK-6.

ATP:citrate lyase [ATP citrate (pro-3S)-lyase; EC 4.1.3.8] was purified and characterized from the cells of Hydrogenobacter thermophilus, an aerobic, thermophilic, hydrogen-oxidizing bacterium which fixes carbon dioxide by a reductive carboxylic acid cycle. The enzyme was quite stable, even in the absence of sulfhydryl reagents. Optimum pH for reaction was 6.7 to 6.9, and optimum temperature was around 80 degrees C. The molecular weight of native enzyme was estimated to be 260,000 by gel filtration analysis, and that of a subunit was estimated to be 43,000 by sodium dodecyl sulfate-polyacrylamide gel analysis. Km values for reaction components were as follows: citrate, 6.25 mM; ATP, 650 microM; coenzyme A, 40.8 microM; and Mg2+, 8 mM. The enzyme showed citrate synthase activity in the presence of Mg2+, but the reaction rate was very low (less than 1/200 of the lyase activity).     

Characterization of ATP citrate lyase from Chlorobium limicola.

ATP citrate lyase (EC 4.1.3.8) from Chlorobium limicola was partially purified. It was established that the consumption of substrates and the formation of products proceeded stoichiometrically and that citrate cleavage was of the si-type. ADP and oxaloacetate inhibited enzyme activity. Oxaloacetate also inhibited the growth of C. limicola.     

Heterogeneous distribution of ATP citrate lyase in rat-liver parenchyma. Microradiochemical determination in microdissected periportal and perivenous liver tissue

1. A radiochemical microtest was established for the determination of ATP citrate lyase in tissue samples of 0.2-1.0 micrograms dry weight. The specificity of this test system was guaranteed by its coenzyme A dependence as well as by inhibition of the activity measured in presence of a specific antibody. 2. Using this test system ATP citrate lyase activity was determined in microdissected periportal and perivenous liver tissue of fed, fasted and refed animals. The perivenous activity was 1.8-fold and 2.4-fold higher than the periportal one in fed male and female rats respectively. 3. The perivenous to periportal gradient was decreased during starvation-dependent reduction of the ATP citrate lyase activity. On the other hand it was not only restored but enhanced up to 2.8 after refeeding-dependent enhancement of the enzyme activity. 4. The predominance of the ATP citrate lyase activity in the perivenous, mainly glycolytic zone supports the hypothesis of the coordinate zonation of the carbohydrate and the lipid metabolism in the liver parenchyma.     

Fructose-1,6-bisphosphatase from rat liver. A comparison of the kinetics of the unphosphorylated enzyme and the enzyme phosphorylated by cyclic AMP-dependent protein kinase

A purification procedure for rat hepatic fructose-1,6-bisphosphatase, described earlier, has been improved, resulting in an enzyme preparation with a neutral pH optimum and with both phosphorylatable serine residues present. The subunit Mr was 40,000. Phosphorylation in vitro with cyclic AMP-dependent protein kinase resulted in the incorporation of 1.4 mol of phosphate/mol of subunit and led to an almost 2-fold decrease in apparent Km for fructose-1,6-bisphosphate. In contrast to yeast fructose-1,6-bisphosphatase, fructose-2,6-bisphosphate had no effect on the rate of phosphorylation or dephosphorylation of the intact enzyme. The effects of the composition of the assay medium, with regard to buffering substance and Mg2+ concentration, on the apparent Km values of phosphorylated and unphosphorylated enzyme were investigated. The kinetics of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase were studied with special reference to the inhibitory effects of adenine nucleotides and fructose-2,6-bisphosphate. Unphosphorylated fructose-1,6-bisphosphatase was more susceptible to inhibition by both AMP and fructose 2,6-bisphosphate than phosphorylated enzyme, at high and low substrate concentrations. Both ATP and ADP had a similar effect on the two enzyme forms, ADP being the more potent inhibitor. Finally, the combined effect of several inhibitors at physiological concentrations was studied. Under conditions resembling the gluconeogenic state, phosphorylated fructose-1,6-bisphosphatase was found to have twice the activity of the unphosphorylated enzyme.     

Presence and regulation of ATP:citrate lyase from the citric acid producing fungus Aspergillus niger

ATP:citrate lyase (EC 4.1.3.8) has been identified in cell-free extracts from the filamentous fungus Aspergillus niger. The enzyme was located in the cytosol. It exhibits an activity at least ten times that of acetate-CoA-kinase (EC 6.2.1.1) during growth on carbohydrates as carbon sources, and is thus considered responsible for acetyl-CoA formation under these conditions. It is formed constitutively and its biosynthesis does not appear to be controlled by changes in the nitrogen or carbon source or type. ATP:citrate-lyase appears to be very labile during conventional purification procedures; a method involving fast protein liquid anion exchange chromatography was thus developed in order to obtain enzyme preparations sufficiently free of enzymes which could interfere with kinetic investigations. This preparation displays commonly known characteristics of ATP:citrate lyase with respect to substrate affinities and cofactor requirements, with the exception that the affinity for citrate is rather low (2.5 mM). No activator was found. The enzyme is inhibited by nucleoside diphosphates, nucleoside monophosphates and palmitoyl-CoA. Regulation of ATP:citrate lyase be the energy charge of the cytosol in relation to lipid or citric acid accumulation is discussed in view of these findings. Present address: Institut für Allgemeine Biochemie, Universität Wien, Währingerstrasse 38, A-1090 Wien, Austria