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[27] ATP citrate lyase (citrate-cleavage enzyme)
[27]
ATP CITRATE LYASE
153
[27] A T P C i t r a t e L y a s e ( C i t r a t e - C l e a v a g e E n z y m e ) [EC 4.1.3.8
ATP: citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephosphorylating)]
By YOSHIRO TAKEDA, FUJIO SUZUKI, a n d HIDEO INOUE Mg++
Citrate -t- ATP -t- CoA ~
acetyl-CoA -~ oxaloacetate -t~ ADP + P,
Assay Methods Principle. ATP citrate lyase is assayed by determining the amount of acetyl-CoA or oxaloacetate formed. Three" methods have been employed. (1) The hydroxamate method: The acetyl-CoA formed is trapped as acetylhydroxamate and the latter is determined by the color produced with FeCls. 1 (2) The spectrophotometric method: The oxaloacetate formed is measured by its reaction with NADH in the presence of malate dehydrogenase.2 (3) The isotopic method: Citrate-l,5-1'C is incubated with ATP, CoA, Mg ÷÷, and enzyme, and the oxaloacetate-14C formed is degraded according to the method of Krebs and Eggleston2 The ~4C02 evolved is trapped and counted.', 5 The hydroxamate method is applicable to only a narrow range of enzyme concentrations, but it is useful in purification steps 1 through 3 because cruder preparations contain a significant activity of endogenous NADH oxidation which sometimes interferes with the use of the spectrophotometric method. The spectrophotometric method is used in the subsequent steps of purification {steps 4 through 7). Because of its high sensitivity, the isotopic method is employed when the ATP citrate lyase activity in some tissue is so low that the methods (1) and (2) cannot be applied. It can also be used when one of the reaction products, acetyl-CoA or oxaloacetate, is present in the reaction system. The Hydroxamate Method Reagents
Tris buffer, 0.5 M, pH 8.4 MgCI2, 0.2 M l p. A. Srere and F. Lipmann, J. Am. Chem. Soc. 75, 4874 (1953). See also Vol. V
[ss]. s p. A. Srere, J. Biol. Chem. ~
2544 (1959).
' H. A. Krebs and L. V. Eggleston, Biochem. J. 39, 408 (1945). H. Inoue, F. Suzuki, K. Fukunishi, K. Adachi, and Y. Takeda, J. Biochem. 60, 543
(1966). F. Suzuki, K. Fukunishi, Y. Daikuhara, and Y. Takeda, J. Biochem. 62, 170 (1967).
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REACTIONS LEADING TO AND FROM THE CYCLE
[27]
2-Mercaptoethanol, 0.2 M Potassium citrate, 0.2 M CoA, 2 mM Hydroxylaminc, 2 M, adjusted to pH 8.4 with KOH ATP, 0.1 M Trichloroacetic acid, 20% FeC13, 2 M
Procedure. The following components are added: Tris buffer, 0.4 m]; MgCl:, 0.05 ml; 2-mercaptoethanol, 0.05 ml; potassium citrate, 0.1 ml; CoA, 0.05 ml; hydroxylamine, 0.1 ml; enzyme to be assayed; and water to make a total volume of 0.95 ml. A blank tube is prepared with all components except CoA. The reaction is started by adding 0.05 ml of ATP. After 30 minutes at 37 °, 1.2 ml of trichloroacetic acid and then 0.3 ml of FeC13 are added; the acetylhydroxamate formed is measured at 520 mt~. The Spectrophotometric Method Reagents Tris buffer, 0.5 M, pH 8.4 MgCl~, 0.2 M 2-Mercaptoethanol, 0.2 M Potassium citrate, 0.2 M CoA, 2 mM ATP, 0.1 M NADH, l0 mM Malate dehydrogenase, 20 units/ml
Procedure. The following components are added to a 1.5 ml silica cell (length = 1 cm): Tris buffer, 0.4 ml; MgCl~, 0.05 ml; 2-mercaptoethanol, 0.05 ml; potassium citrate, 0.1 ml; ATP, 0.1 ml; NADH, 0.02 ml; malate dehydrogenase, 0.01 ml; enzyme to be assayed; and water to make a total volume of 0.90 ml. A blank cell is prepared with all components except ATP and CoA. The reaction is started by adding 0.1 ml of CoA. The decrease in absorption at 340 n ~ at 37 ° is measured. The Isotopic Method Reagents Tris buffer, 0.2 M, pH 8.4 MgCI2, 0.2 M 2-Mercaptoethanol, 0.2 M
[27]
ATP CITR&TE LYASE
155
Potassium citrate-l,5-~4C (0.5 ~C per micromole), 20 mM CoA, 2 mM ATP, 0.1 M Oxaloacetic acid, 0.1 M, in 2 N HC1 Phthalate buffer, 0.75 M: 15.3 g of potassium hydrogen phthalate and 1.8 g of N a 0 H in 100 ml of water A12(SO4)~" 18 H20, 33.3~'o Hyamine, 1 M, in methanol Procedure. Incubation is carried out in a double-armed Warburg flask equipped with a serum bottle stopper. The flask contains Tris buffer, 0.2 ml; MgC12, 0.05 ml; 2-mereaptoethanol, 0.05 ml; CoA, 0.05 ml; potassium citrate-l,5-14C, 0.1 ml; enzyme to be assayed; and water to make a total volume of 0.90 ml in the main compartment, and 0.1 ml of ATP in a side arm. A blank flask is prepared with all components except CoA. The reaction is started by adding ATP. After incubation for an appropriate time at 37 °, the reaction is stopped by addition of 0.2 ml of oxaloacetic acid in 2 N HC1. For the degradation of oxaloacetic acid, 0.5 ml of phthalate buffer is placed in one side arm, 0.5 ml of aluminum sulfate solution in the other, and 0.2 ml of methanotic hyamine solution in the center well. After equilibration for 10 minutes at 25 °, the tap is closed and the phthalate buffer is introduced from the side arm followed by the aluminum sulfate solution. The flask is shaken for another 75 minutes at 25 °. The 1~C02 evolved is trapped by absorption on methanolic hyamine solution in the center well. Then the hyamine solution is transferred to scintillator-toluene solution with 0.5 ml of methanol. Radioactivities are measured in a liquid scintillation spectrometer. Protein Determination. Protein concentrations are determined by the biuret method 6 in the crude extract and by the procedure of Lowry et al. 7 in the subsequent steps. Units. One unit of enzyme is defined as the amount of enzyme that forms 1 micromole of acetylhydroxamate, oxidizes 1 micromole of NADH, or evolves 1 micromolc of 1~C02 per minute at 37 °.
Purification Procedure Treatment of Animals. Adult albino rats are pretreated with a highsucrose diet in order to induce ATP citrate lyase in the liver. The animals are starved for 2 days and then fed on a high-sucrose diet for 8A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949). See also Vol. III, p. 450. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). See also Vol. III, p. 448.
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[27]
3 days before being killed.'. 8 The high-sucrose diet contains 63% sucrose, 30% casein, 4% salt mixture, 2% cellulose powder, l~b vitamin mixture, and 0.1% choline chloride. This diet results in about 10-fold the normal level of the enzyme in the liver. Step 1. Preparation o] the Crude Extract. All operations are carried out at 0-4 °. Fresh livers (550 g), from rats which have been fed on a high-sucrose diet, are homogenized in 4 volumes of 0.25 M sucrose containing 20 mM Tris buffer (pH 8.0) with the aid of a PotterElvehjem glass homogenizer. The homogenate is centrifuged at 15,000 g for 30 minutes and then 105,000 g for 30 minutes. To the supernatant fluid thus obtained (crude extract), 1 M Tris buffer (pH 8.0) is added to adjust the pH to about 7.6 and then 2-mereaptoethanol and MgCI2 are added to give final concentrations of 10 mM and 1 raM, respectively, in order to stabilize the enzyme. All subsequent steps are performed in the presence of these two agents. Step ~. Ammonium Sulfate Fractionation. To the crude extract, ammonium sulfate is added to 25% saturation. The precipitate formed is removed by centrifugation and discarded. Further ammonium sulfate is then added to the supernatant to 45~ saturation. The resulting precipitate is collected by eentrifugation and dissolved in 0.01 M Tris buffer (pH 7.8). The enzyme solution is dialyzed overnight against 30 volumes of the same buffer. Step 3. DEAE-Cellulose Column Chromatography. The dialyzed enzyme solution containing 25.3 g of protein is diluted with 0.01 M Tris buffer (pH 7.8), to give 20 mg of protein per milliliter, and then applied to a DEAE-cellulose column (Sigma, medium mesh; 8 cm in diameter and 35 cm in height), equilibrated with 0.005 M Tris buffer (pH 7.8). Elution is effected by using a continuous gradient (convex shape) of KC1 in 0.005 M Tris buffer (pH 7.4) with an initial concentration of 0.02 M in the mixing chamber (3 liters) and 0.4 M in the reservoir (2 liters). The eluate is collected in 20-ml fractions in tubes containing 1 ml of 1 M Tris buffer (pH 7.4) at a flow rate of 4 ml per minute. The enzyme activity is usually eluted between 2000 and 2500 ml. Fractions having a specific activity of over 0.4 are pooled and concentrated with ammonium sulfate at 40% saturation. After centrifugation, the precipitate is dissolved in a small volume of 0.005 M Tris buffer (pH 7.4) and dialyzed overnight against 200 volumes of the same buffer. Step ~. Alumina C~ Gel Treatment. Step 3 enzyme is adjusted to pH 6.8 with 1 M acetate buffer (pH 5.0) and then mixed with an amount of alumina Cv gel equivalent to that of the protein. The mixture is a M. S. Kornacker and J. M. Lowenstein, Biochem. J. 94, 209 (1965); ibid. 95, 832
(1965).
[27]
ATP CITRATE LYASE
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stirred for 15 minutes, then centrifuged; supernatant fluid is discarded. The precipitate is washed twice with 200 ml portions of 10 mM potassium citrate (pH 7.4), and then the enzyme is eluted successively with three 200 ml portions of 0.15M and finally with 200 ml of 0.3M potassium citrate (pH 7.4). The eluates obtained with 0.15 M and 0.3 M potassium citrate are combined and concentrated with ammonium sulfate at 40% saturation. After centrifugation, the precipitate is taken up in a small volume of 5 mM Tris buffer (pH 7.4) and dialyzed overnight against 200 volumes of the same buffer. Step 5. Brushite Column Chromatography. The dialyzed enzyme of step 4 is applied to a column of brushite (1 cm 3 of brushite per milligram of protein) equilibrated with 5 mM potassium phosphate buffer (pH 7.4). After washing the column with a bed volume of the same buffer, elution is carried out with 70 mM potassium phosphate buffer (pH 7.4) at a flow rate of about 1 ml per minute. Fractions having a specific activity of over 3.8 are collected. Step 6. First Gel Filtration on Sephadex G-200. Step 5 enzyme is precipitated by addition of ammonium sulfate to 40% saturation and dissolved in a small volume of 10 mM Tris buffer (pH 7.4) containing 50 mM potassium citrate (pH 7.4). Then the enzyme is applied to a column of heat-treated Sephadex G-2009 (equivalent to 50 volumes of the enzyme solution), which has been equilibrated with the same buffer. The column is eluted with the same buffer in 3 ml fractions at a flow rate of about 15 ml per hour. The elution pattern of protein usually gives two peaks. The first is the major peak and the second is a minor peak. The enzyme activity is associated with the first major peak, and colored impurities, if any, with the second minor peak. Active fractions, having a specific activity of over 5.5, are combined. Step 7. Second Gel Filtration on Sephadex G-200. The combined active fractions of step 6 are concentrated by treatment with 40% saturation of ammonium sulfate and then subjected to a second gel filtration treatment on Sephadex G-200 in the same way as described in step 6. Active fractions with the same specific activity (6.25) are collected and combined. The purification of the enzyme is summarized in the table. Comments. During the course of purification, some variations have been encountered in the brushite step. The separation of the enzyme and colored impurities (brownish yellow) in this step is satisfactory in most ' S e p h a d e x G-200 (Pharmaeia) is suspended in distilled water and boiled for 30 minutes. Then, the particles are w:(shed 20 times by decantation with warm distilled water at 60 ° to remove fine material and stored in 10 m M Tris buffer (pH 7.4) containing 50 m M potassium citrate ( p H 7.4) and 1 m M MgCl~.
158
[27]
REACTIONS LEADING TO AND FROM THE CYCLE SUMMARY OF PURIFICATION PROCEDURE
Step 1. Crude extract 2. Ammonium sulfate fractionation 3. DEAE-cellulose column chromatography 4. Alumina C~ gel treatment 5. Brushite column chromatography 6. First gel filtration on Sephadex G-200 7. Second gel filtration on Sephadex G-200
Protein (rag)
Total activity (units) °
56,100 25,300
6,452 5,060
0.12 0.20
100 78.4
3,210
4,350
1.36
67.4
2,040
4,202
2.06
65.1
541
2,299
4.25
35.6
288
1,713
5.95
26.5
220
1,375
6.25 b
21.3
Specific activity
Recovery (%)
o See section on units under assay methods. b This value is slightly higher than that previously reported (see text footnote 4). However, the dilution factor of the enzyme at the last step is so great that the highest specific activities vary from 5.9 to 6.3 under the assay conditions. cases, although on some occasions the enzyme is retained more tightly on the column and is eluted not with 70 mM, but with 0.3 M, potassium phosphate buffer (pH 7.4). In this case, there is more trailing of the enzyme peak and a higher percentage of its activity, up to 3 0 - 4 0 ~ , is eluted with the colored impurities. The reason for this is not clear, but these impurities can be removed in the subsequent step, step 6. The DEAE-cellulose and the Sephadex G-200 column steps are highly reproducible. Crystallization. Crystallization of the enzyme is carried out as follows. Step 7 enzyme is precipitated by addition of ammonium sulfate to 5 0 ~ saturation. After centrifugation, the precipitate is extracted successively with 40, 35, 30, and 25% ammonium sulfate solutions (3 ml each) containing 10 m M Tris buffer (pH 7.4), 50 m M potassium citrate (pH 7.4), 10 m M 2-mercaptoethanol and 1 m M MgCl~. Each extract is stored at 4 ° . After several days, small colorless plates appear in the extract with 30% ammonium sulfate. The specific activity of the enzyme remains the same after crystallization. Properties
Purity. The purified enzyme moves as a single homogeneous protein on sedimentation and on moving boundary electrophoresis. The homo-
[27]
ATP CITRATE LYASE
159
geneity of the enzyme is also demonstrated by immunological criteria. The purified enzyme contains no chromophore and is also free of nucleotides and nucleic acids. Assays for citrate synthase, isocitrate, dehydrogenase (NADP), malate dehydrogenase, aconitate hydratase, reduced NADP dehydrogenase, adenylate kinase, acetyl-CoA carboxylase, and fatty acid synthase are all negative. Molecular Weight. The sedimentation coefficient calculated for water at 20 ° and zero protein concentration is 13.5 S and the diffusion constant (D2o, w) is found to be 2.62 X 10-7 cm2/sec. From these values, the molecular weight of the enzyme is calculated to be approximately 500,000, assuming a partial specific volume of 0.75. Specificity. The enzyme is highly specific for citrate. Other tricarboxylic acids tested, such as tricarballylate, c/s- and trans-aconitates and DL-isocitrate, are not attacked at any detectable rate. The enzyme is specific also for ATP, and other triphosphonucleotides tested, such as CTP, GTP, and UTP, have little or no effect. The activity of this enzyme depends on the presence of Mg +÷. Mn ++ can partially substitute for Mg +÷, but Ni ÷÷, Fe ÷÷, Fe ~÷, Cu *+ and Zn +÷ are all inactive. The enzyme requires sulfhydryl compounds for maximal activity. 2-Mercaptoethanol, cysteine, glutathione, and dithiothreitol are all nearly equally effective. Stability. The purified enzyme preparation is labile on storage and retains only about a quarter of its activity after 3 days and one-tenth after 5 days when stored under air at 3 ° in 10 mM Tris buffer (pH 7.8). The inactivation of the enzyme can be effectively prevented by the addition of sulfhydryl compounds, such as 2-mercaptoethanol, glutathione, or cysteine, together with MgC12. The most satisfactory method for storage is the use of dithiothreitol. In the presence of this reagent at a concentration of 10 mM together with 1 mM MgCl~, the enzyme is quite stable at 3 ° under an atmosphere of nitrogen for at least 2 weeks. Freezing of the enzyme in the presence of sulfhydryl compounds is not recommended for storage. pH Optimum. With Tris buffer, maximal activity is obtained at about pH 8.4. At pH 7.0 and 9.0, the activity is about 60% of that seen at pH 8.4. K,~ Values. The Km values for citrate and ATP at pH 8.4 are found to be 0.56 mM and 0.172 raM, respectively. Inhibitors. ADP, one of the reaction products, inhibits the reaction competitively with respect to ATP. The Ki value for ADP at pH 8.4 is 0.172 mM. Inorganic orthophosphate at a concentration of 20 mM lowers the enzyme activity to about 30% of the control value. Distribution. ATP citrate lyase is widely distributed in the soluble
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REACTIONS LEADING TO AND FROM THE CYCLE
[28]
fraction of a variety of animal tissues, e.g., liver, brain, heart, kidney, and adipose tissue; it is found also in certain bacteria. Organ and Species ~pecificities. The enzymes from rat tissues, e.g., kidney, heart, brain, and adipose tissue, cannot be distinguished from rat liver enzyme on the basis of their reactivity with rat liver antienzyme, whereas the enzymes from the livers of chick, dog, guinea pig, and rabbit react only partially with the same antienzyme.
[28] Citrate Lyase [EC 4.1.3.6
Citrate oxaloacetate-lyase]
By S. "[')AGLEY Citrate ~ oxaloacetate -{- acetate The preparation of citrate lyase from Klebsiella aerogenes (Aerobacter aerogenes) described previously 1 has been improved to provide an enzyme that sediments with a single symmetrical peak having an S2o., value of 16 S in the analytical ultracentrifuge? The purified enzyme, like cruder preparations, 3 is activated by a range of divalent metal ions (Mg ÷÷, Mn ++, Fe ++, and Zn÷+), shows optimal activity at pH 8.0-9.0, and is powerfully inhibited by oxaloacetate. 4 The keto form of this compound is a substrate for the enzyme, but the enol form is not and may be responsible for the inhibition observed2 On account of this inhibition, true equilibrium can be reached in the direction of citrate cleavage only when the initial concentration of citrate does not exceed 2 mM and when enzyme, Mg ++, and acetate are provided in excess2 Apparent equilibrium constants expressed in terms of total citrate, oxaloacetate, and acetate concentrations are markedly affected by the initial concentrations of the reactants2 This is due to the ability of Mg *÷ ions, added as cofactor, to complex with citrate and the isomers of oxaloacetate. For this reason, and also because Mg ~ and oxaloacetate complex, respectively, with phosphate and Tris which were used as buffers, earlier values of equilibrium constants 1,~ appear to be in error, e When the reaction is performed in triethanolamine-hydrochloric acid buffer, pH 8.4, which does
1H. H. Daron and I. C. Gunsalus, Vol. V [85]. ' C. Siva Raman, Bioehim. Biophys. Aeta 52, 212 (1961). sS. Dagley and E. A. Dawes, Biochlm. Biophys. Aeta 17, 177 (1955). T. J. Bowen and L. J. Rogers, Biochim. Biophys. Aeta 77, 685 (1963). 'S. S. Tate and S. P. Datta, Bioehem. J, 911 18c (1964). 'S. S. Tate and S. P. Datta, Biochem. J. 94, 470 (1965). 'R. J. Harvey and E. B. Collins, J. Biol. Chem. 238, 2648 (1963).
ATP CITRATE LYASE
153
[27] A T P C i t r a t e L y a s e ( C i t r a t e - C l e a v a g e E n z y m e ) [EC 4.1.3.8
ATP: citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephosphorylating)]
By YOSHIRO TAKEDA, FUJIO SUZUKI, a n d HIDEO INOUE Mg++
Citrate -t- ATP -t- CoA ~
acetyl-CoA -~ oxaloacetate -t~ ADP + P,
Assay Methods Principle. ATP citrate lyase is assayed by determining the amount of acetyl-CoA or oxaloacetate formed. Three" methods have been employed. (1) The hydroxamate method: The acetyl-CoA formed is trapped as acetylhydroxamate and the latter is determined by the color produced with FeCls. 1 (2) The spectrophotometric method: The oxaloacetate formed is measured by its reaction with NADH in the presence of malate dehydrogenase.2 (3) The isotopic method: Citrate-l,5-1'C is incubated with ATP, CoA, Mg ÷÷, and enzyme, and the oxaloacetate-14C formed is degraded according to the method of Krebs and Eggleston2 The ~4C02 evolved is trapped and counted.', 5 The hydroxamate method is applicable to only a narrow range of enzyme concentrations, but it is useful in purification steps 1 through 3 because cruder preparations contain a significant activity of endogenous NADH oxidation which sometimes interferes with the use of the spectrophotometric method. The spectrophotometric method is used in the subsequent steps of purification {steps 4 through 7). Because of its high sensitivity, the isotopic method is employed when the ATP citrate lyase activity in some tissue is so low that the methods (1) and (2) cannot be applied. It can also be used when one of the reaction products, acetyl-CoA or oxaloacetate, is present in the reaction system. The Hydroxamate Method Reagents
Tris buffer, 0.5 M, pH 8.4 MgCI2, 0.2 M l p. A. Srere and F. Lipmann, J. Am. Chem. Soc. 75, 4874 (1953). See also Vol. V
[ss]. s p. A. Srere, J. Biol. Chem. ~
2544 (1959).
' H. A. Krebs and L. V. Eggleston, Biochem. J. 39, 408 (1945). H. Inoue, F. Suzuki, K. Fukunishi, K. Adachi, and Y. Takeda, J. Biochem. 60, 543
(1966). F. Suzuki, K. Fukunishi, Y. Daikuhara, and Y. Takeda, J. Biochem. 62, 170 (1967).
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REACTIONS LEADING TO AND FROM THE CYCLE
[27]
2-Mercaptoethanol, 0.2 M Potassium citrate, 0.2 M CoA, 2 mM Hydroxylaminc, 2 M, adjusted to pH 8.4 with KOH ATP, 0.1 M Trichloroacetic acid, 20% FeC13, 2 M
Procedure. The following components are added: Tris buffer, 0.4 m]; MgCl:, 0.05 ml; 2-mercaptoethanol, 0.05 ml; potassium citrate, 0.1 ml; CoA, 0.05 ml; hydroxylamine, 0.1 ml; enzyme to be assayed; and water to make a total volume of 0.95 ml. A blank tube is prepared with all components except CoA. The reaction is started by adding 0.05 ml of ATP. After 30 minutes at 37 °, 1.2 ml of trichloroacetic acid and then 0.3 ml of FeC13 are added; the acetylhydroxamate formed is measured at 520 mt~. The Spectrophotometric Method Reagents Tris buffer, 0.5 M, pH 8.4 MgCl~, 0.2 M 2-Mercaptoethanol, 0.2 M Potassium citrate, 0.2 M CoA, 2 mM ATP, 0.1 M NADH, l0 mM Malate dehydrogenase, 20 units/ml
Procedure. The following components are added to a 1.5 ml silica cell (length = 1 cm): Tris buffer, 0.4 ml; MgCl~, 0.05 ml; 2-mercaptoethanol, 0.05 ml; potassium citrate, 0.1 ml; ATP, 0.1 ml; NADH, 0.02 ml; malate dehydrogenase, 0.01 ml; enzyme to be assayed; and water to make a total volume of 0.90 ml. A blank cell is prepared with all components except ATP and CoA. The reaction is started by adding 0.1 ml of CoA. The decrease in absorption at 340 n ~ at 37 ° is measured. The Isotopic Method Reagents Tris buffer, 0.2 M, pH 8.4 MgCI2, 0.2 M 2-Mercaptoethanol, 0.2 M
[27]
ATP CITR&TE LYASE
155
Potassium citrate-l,5-~4C (0.5 ~C per micromole), 20 mM CoA, 2 mM ATP, 0.1 M Oxaloacetic acid, 0.1 M, in 2 N HC1 Phthalate buffer, 0.75 M: 15.3 g of potassium hydrogen phthalate and 1.8 g of N a 0 H in 100 ml of water A12(SO4)~" 18 H20, 33.3~'o Hyamine, 1 M, in methanol Procedure. Incubation is carried out in a double-armed Warburg flask equipped with a serum bottle stopper. The flask contains Tris buffer, 0.2 ml; MgC12, 0.05 ml; 2-mereaptoethanol, 0.05 ml; CoA, 0.05 ml; potassium citrate-l,5-14C, 0.1 ml; enzyme to be assayed; and water to make a total volume of 0.90 ml in the main compartment, and 0.1 ml of ATP in a side arm. A blank flask is prepared with all components except CoA. The reaction is started by adding ATP. After incubation for an appropriate time at 37 °, the reaction is stopped by addition of 0.2 ml of oxaloacetic acid in 2 N HC1. For the degradation of oxaloacetic acid, 0.5 ml of phthalate buffer is placed in one side arm, 0.5 ml of aluminum sulfate solution in the other, and 0.2 ml of methanotic hyamine solution in the center well. After equilibration for 10 minutes at 25 °, the tap is closed and the phthalate buffer is introduced from the side arm followed by the aluminum sulfate solution. The flask is shaken for another 75 minutes at 25 °. The 1~C02 evolved is trapped by absorption on methanolic hyamine solution in the center well. Then the hyamine solution is transferred to scintillator-toluene solution with 0.5 ml of methanol. Radioactivities are measured in a liquid scintillation spectrometer. Protein Determination. Protein concentrations are determined by the biuret method 6 in the crude extract and by the procedure of Lowry et al. 7 in the subsequent steps. Units. One unit of enzyme is defined as the amount of enzyme that forms 1 micromole of acetylhydroxamate, oxidizes 1 micromole of NADH, or evolves 1 micromolc of 1~C02 per minute at 37 °.
Purification Procedure Treatment of Animals. Adult albino rats are pretreated with a highsucrose diet in order to induce ATP citrate lyase in the liver. The animals are starved for 2 days and then fed on a high-sucrose diet for 8A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949). See also Vol. III, p. 450. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). See also Vol. III, p. 448.
156
REACTIONS LEADING TO AND FROM THE CYCLE
[27]
3 days before being killed.'. 8 The high-sucrose diet contains 63% sucrose, 30% casein, 4% salt mixture, 2% cellulose powder, l~b vitamin mixture, and 0.1% choline chloride. This diet results in about 10-fold the normal level of the enzyme in the liver. Step 1. Preparation o] the Crude Extract. All operations are carried out at 0-4 °. Fresh livers (550 g), from rats which have been fed on a high-sucrose diet, are homogenized in 4 volumes of 0.25 M sucrose containing 20 mM Tris buffer (pH 8.0) with the aid of a PotterElvehjem glass homogenizer. The homogenate is centrifuged at 15,000 g for 30 minutes and then 105,000 g for 30 minutes. To the supernatant fluid thus obtained (crude extract), 1 M Tris buffer (pH 8.0) is added to adjust the pH to about 7.6 and then 2-mereaptoethanol and MgCI2 are added to give final concentrations of 10 mM and 1 raM, respectively, in order to stabilize the enzyme. All subsequent steps are performed in the presence of these two agents. Step ~. Ammonium Sulfate Fractionation. To the crude extract, ammonium sulfate is added to 25% saturation. The precipitate formed is removed by centrifugation and discarded. Further ammonium sulfate is then added to the supernatant to 45~ saturation. The resulting precipitate is collected by eentrifugation and dissolved in 0.01 M Tris buffer (pH 7.8). The enzyme solution is dialyzed overnight against 30 volumes of the same buffer. Step 3. DEAE-Cellulose Column Chromatography. The dialyzed enzyme solution containing 25.3 g of protein is diluted with 0.01 M Tris buffer (pH 7.8), to give 20 mg of protein per milliliter, and then applied to a DEAE-cellulose column (Sigma, medium mesh; 8 cm in diameter and 35 cm in height), equilibrated with 0.005 M Tris buffer (pH 7.8). Elution is effected by using a continuous gradient (convex shape) of KC1 in 0.005 M Tris buffer (pH 7.4) with an initial concentration of 0.02 M in the mixing chamber (3 liters) and 0.4 M in the reservoir (2 liters). The eluate is collected in 20-ml fractions in tubes containing 1 ml of 1 M Tris buffer (pH 7.4) at a flow rate of 4 ml per minute. The enzyme activity is usually eluted between 2000 and 2500 ml. Fractions having a specific activity of over 0.4 are pooled and concentrated with ammonium sulfate at 40% saturation. After centrifugation, the precipitate is dissolved in a small volume of 0.005 M Tris buffer (pH 7.4) and dialyzed overnight against 200 volumes of the same buffer. Step ~. Alumina C~ Gel Treatment. Step 3 enzyme is adjusted to pH 6.8 with 1 M acetate buffer (pH 5.0) and then mixed with an amount of alumina Cv gel equivalent to that of the protein. The mixture is a M. S. Kornacker and J. M. Lowenstein, Biochem. J. 94, 209 (1965); ibid. 95, 832
(1965).
[27]
ATP CITRATE LYASE
157
stirred for 15 minutes, then centrifuged; supernatant fluid is discarded. The precipitate is washed twice with 200 ml portions of 10 mM potassium citrate (pH 7.4), and then the enzyme is eluted successively with three 200 ml portions of 0.15M and finally with 200 ml of 0.3M potassium citrate (pH 7.4). The eluates obtained with 0.15 M and 0.3 M potassium citrate are combined and concentrated with ammonium sulfate at 40% saturation. After centrifugation, the precipitate is taken up in a small volume of 5 mM Tris buffer (pH 7.4) and dialyzed overnight against 200 volumes of the same buffer. Step 5. Brushite Column Chromatography. The dialyzed enzyme of step 4 is applied to a column of brushite (1 cm 3 of brushite per milligram of protein) equilibrated with 5 mM potassium phosphate buffer (pH 7.4). After washing the column with a bed volume of the same buffer, elution is carried out with 70 mM potassium phosphate buffer (pH 7.4) at a flow rate of about 1 ml per minute. Fractions having a specific activity of over 3.8 are collected. Step 6. First Gel Filtration on Sephadex G-200. Step 5 enzyme is precipitated by addition of ammonium sulfate to 40% saturation and dissolved in a small volume of 10 mM Tris buffer (pH 7.4) containing 50 mM potassium citrate (pH 7.4). Then the enzyme is applied to a column of heat-treated Sephadex G-2009 (equivalent to 50 volumes of the enzyme solution), which has been equilibrated with the same buffer. The column is eluted with the same buffer in 3 ml fractions at a flow rate of about 15 ml per hour. The elution pattern of protein usually gives two peaks. The first is the major peak and the second is a minor peak. The enzyme activity is associated with the first major peak, and colored impurities, if any, with the second minor peak. Active fractions, having a specific activity of over 5.5, are combined. Step 7. Second Gel Filtration on Sephadex G-200. The combined active fractions of step 6 are concentrated by treatment with 40% saturation of ammonium sulfate and then subjected to a second gel filtration treatment on Sephadex G-200 in the same way as described in step 6. Active fractions with the same specific activity (6.25) are collected and combined. The purification of the enzyme is summarized in the table. Comments. During the course of purification, some variations have been encountered in the brushite step. The separation of the enzyme and colored impurities (brownish yellow) in this step is satisfactory in most ' S e p h a d e x G-200 (Pharmaeia) is suspended in distilled water and boiled for 30 minutes. Then, the particles are w:(shed 20 times by decantation with warm distilled water at 60 ° to remove fine material and stored in 10 m M Tris buffer (pH 7.4) containing 50 m M potassium citrate ( p H 7.4) and 1 m M MgCl~.
158
[27]
REACTIONS LEADING TO AND FROM THE CYCLE SUMMARY OF PURIFICATION PROCEDURE
Step 1. Crude extract 2. Ammonium sulfate fractionation 3. DEAE-cellulose column chromatography 4. Alumina C~ gel treatment 5. Brushite column chromatography 6. First gel filtration on Sephadex G-200 7. Second gel filtration on Sephadex G-200
Protein (rag)
Total activity (units) °
56,100 25,300
6,452 5,060
0.12 0.20
100 78.4
3,210
4,350
1.36
67.4
2,040
4,202
2.06
65.1
541
2,299
4.25
35.6
288
1,713
5.95
26.5
220
1,375
6.25 b
21.3
Specific activity
Recovery (%)
o See section on units under assay methods. b This value is slightly higher than that previously reported (see text footnote 4). However, the dilution factor of the enzyme at the last step is so great that the highest specific activities vary from 5.9 to 6.3 under the assay conditions. cases, although on some occasions the enzyme is retained more tightly on the column and is eluted not with 70 mM, but with 0.3 M, potassium phosphate buffer (pH 7.4). In this case, there is more trailing of the enzyme peak and a higher percentage of its activity, up to 3 0 - 4 0 ~ , is eluted with the colored impurities. The reason for this is not clear, but these impurities can be removed in the subsequent step, step 6. The DEAE-cellulose and the Sephadex G-200 column steps are highly reproducible. Crystallization. Crystallization of the enzyme is carried out as follows. Step 7 enzyme is precipitated by addition of ammonium sulfate to 5 0 ~ saturation. After centrifugation, the precipitate is extracted successively with 40, 35, 30, and 25% ammonium sulfate solutions (3 ml each) containing 10 m M Tris buffer (pH 7.4), 50 m M potassium citrate (pH 7.4), 10 m M 2-mercaptoethanol and 1 m M MgCl~. Each extract is stored at 4 ° . After several days, small colorless plates appear in the extract with 30% ammonium sulfate. The specific activity of the enzyme remains the same after crystallization. Properties
Purity. The purified enzyme moves as a single homogeneous protein on sedimentation and on moving boundary electrophoresis. The homo-
[27]
ATP CITRATE LYASE
159
geneity of the enzyme is also demonstrated by immunological criteria. The purified enzyme contains no chromophore and is also free of nucleotides and nucleic acids. Assays for citrate synthase, isocitrate, dehydrogenase (NADP), malate dehydrogenase, aconitate hydratase, reduced NADP dehydrogenase, adenylate kinase, acetyl-CoA carboxylase, and fatty acid synthase are all negative. Molecular Weight. The sedimentation coefficient calculated for water at 20 ° and zero protein concentration is 13.5 S and the diffusion constant (D2o, w) is found to be 2.62 X 10-7 cm2/sec. From these values, the molecular weight of the enzyme is calculated to be approximately 500,000, assuming a partial specific volume of 0.75. Specificity. The enzyme is highly specific for citrate. Other tricarboxylic acids tested, such as tricarballylate, c/s- and trans-aconitates and DL-isocitrate, are not attacked at any detectable rate. The enzyme is specific also for ATP, and other triphosphonucleotides tested, such as CTP, GTP, and UTP, have little or no effect. The activity of this enzyme depends on the presence of Mg +÷. Mn ++ can partially substitute for Mg +÷, but Ni ÷÷, Fe ÷÷, Fe ~÷, Cu *+ and Zn +÷ are all inactive. The enzyme requires sulfhydryl compounds for maximal activity. 2-Mercaptoethanol, cysteine, glutathione, and dithiothreitol are all nearly equally effective. Stability. The purified enzyme preparation is labile on storage and retains only about a quarter of its activity after 3 days and one-tenth after 5 days when stored under air at 3 ° in 10 mM Tris buffer (pH 7.8). The inactivation of the enzyme can be effectively prevented by the addition of sulfhydryl compounds, such as 2-mercaptoethanol, glutathione, or cysteine, together with MgC12. The most satisfactory method for storage is the use of dithiothreitol. In the presence of this reagent at a concentration of 10 mM together with 1 mM MgCl~, the enzyme is quite stable at 3 ° under an atmosphere of nitrogen for at least 2 weeks. Freezing of the enzyme in the presence of sulfhydryl compounds is not recommended for storage. pH Optimum. With Tris buffer, maximal activity is obtained at about pH 8.4. At pH 7.0 and 9.0, the activity is about 60% of that seen at pH 8.4. K,~ Values. The Km values for citrate and ATP at pH 8.4 are found to be 0.56 mM and 0.172 raM, respectively. Inhibitors. ADP, one of the reaction products, inhibits the reaction competitively with respect to ATP. The Ki value for ADP at pH 8.4 is 0.172 mM. Inorganic orthophosphate at a concentration of 20 mM lowers the enzyme activity to about 30% of the control value. Distribution. ATP citrate lyase is widely distributed in the soluble
160
REACTIONS LEADING TO AND FROM THE CYCLE
[28]
fraction of a variety of animal tissues, e.g., liver, brain, heart, kidney, and adipose tissue; it is found also in certain bacteria. Organ and Species ~pecificities. The enzymes from rat tissues, e.g., kidney, heart, brain, and adipose tissue, cannot be distinguished from rat liver enzyme on the basis of their reactivity with rat liver antienzyme, whereas the enzymes from the livers of chick, dog, guinea pig, and rabbit react only partially with the same antienzyme.
[28] Citrate Lyase [EC 4.1.3.6
Citrate oxaloacetate-lyase]
By S. "[')AGLEY Citrate ~ oxaloacetate -{- acetate The preparation of citrate lyase from Klebsiella aerogenes (Aerobacter aerogenes) described previously 1 has been improved to provide an enzyme that sediments with a single symmetrical peak having an S2o., value of 16 S in the analytical ultracentrifuge? The purified enzyme, like cruder preparations, 3 is activated by a range of divalent metal ions (Mg ÷÷, Mn ++, Fe ++, and Zn÷+), shows optimal activity at pH 8.0-9.0, and is powerfully inhibited by oxaloacetate. 4 The keto form of this compound is a substrate for the enzyme, but the enol form is not and may be responsible for the inhibition observed2 On account of this inhibition, true equilibrium can be reached in the direction of citrate cleavage only when the initial concentration of citrate does not exceed 2 mM and when enzyme, Mg ++, and acetate are provided in excess2 Apparent equilibrium constants expressed in terms of total citrate, oxaloacetate, and acetate concentrations are markedly affected by the initial concentrations of the reactants2 This is due to the ability of Mg *÷ ions, added as cofactor, to complex with citrate and the isomers of oxaloacetate. For this reason, and also because Mg ~ and oxaloacetate complex, respectively, with phosphate and Tris which were used as buffers, earlier values of equilibrium constants 1,~ appear to be in error, e When the reaction is performed in triethanolamine-hydrochloric acid buffer, pH 8.4, which does
1H. H. Daron and I. C. Gunsalus, Vol. V [85]. ' C. Siva Raman, Bioehim. Biophys. Aeta 52, 212 (1961). sS. Dagley and E. A. Dawes, Biochlm. Biophys. Aeta 17, 177 (1955). T. J. Bowen and L. J. Rogers, Biochim. Biophys. Aeta 77, 685 (1963). 'S. S. Tate and S. P. Datta, Bioehem. J, 911 18c (1964). 'S. S. Tate and S. P. Datta, Biochem. J. 94, 470 (1965). 'R. J. Harvey and E. B. Collins, J. Biol. Chem. 238, 2648 (1963).