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[12] Cystidine triphosphate synthetase
[12]
CYTIDINE TRIPHOSPHATE SYNTHETASE
79
which is close to the value (7-8 pM) reported for the enzyme from brewer' s yeast. 15
Inhibitors. The enzyme is 70-100% inhibited by p-chloromercuribenzoate, 5,5'-dithiobis(2-nitrobenzoate), or Hg ~+ at 10 pAL but is not significantly inhibited by iodoacetate and iodoacetamide at 1 mM. GMP, GDP, CMP, and CDP are not much inhibitory at 0.5-1 mM. (Strong inhibitions observed previously 1 must have been due to some technical errors.) Uridine nucleotides and EDTA show no inhibition in the standard assay. 15 R. E. H a n d s c h u m a c h e r , J. Biol. Chem. 235, 2917 (1960).
[12] C y t i d i n e T r i p h o s p h a t e
Synthetase
By CEDRIC LONG and DANIEL E. KOSHLAND, JR. Cytidine triphosphate synthetase (CTPS) was initially characterized from Escherichia coli by Lieberman 1 and shown to catalyze the formation of CTP from UTP, ATP, magnesium, and ammonia. A similar reaction was later demonstrated in mammalian tissues 2 and bacteria 3 which used L-glutamine as an amino donor and required a guanosine nucleotide for maximum activities. Following these observations the enzyme was extensively purified from E. coli, and a kinetic analysis revealed a high degree of cooperativity in substrate binding to the enzyme. 4 Thus, both UTP and ATP showed strong site-site interactions, and GTP was found to be an allosteric effector for the glutamine reaction. Part of the high positive cooperativity was shown to proceed through conformational changes involving substrate-induced dimertetramer aggregation. 5 Such features have made CTPS an intriguing model for regulatory proteins. Assay Method
Principle. The enzyme assay measures the amination of UTP spectrophotometrically at 291 nm where UTP has an extinction coefficient I I. L i e b e r m a n , J. Biol. Chem. 222, 675 (1956). 2 H. O. K a m m e n and R. B. Hurlbert, Cancer Res. 19, 654 (1959). a K. B. Chakraborty and R. B. Hurlbert, Biochim. Biophys. Acta 4 7 , 6 0 7 (1961). 4 C. W. L o n g a n d A. B. Pardee, J. Biol. Chem. 242, 4715 (1967). C. W. Long, A. Levitzki, and D. E. K o s h l a n d , Jr., J. Biol. Chem. 245, 80 (1970).
M E T H O D S IN E N Z Y M O L O G Y , VOL. LI
Copyright © 1978by AcademicPress. Inc. All rights of reproduction in any form reserved. 1SBN 0-12-181951-5
80
De Novo
PYRIMID1NE BIOSYNTHESIS
[12]
of 182 and CTP 1520 at pH 7.0. The assay can be carried out with either L-glutamine or ammonia serving as the amino group donor.
Reagents L-glutamine reaction mixture (total of 1.0 ml) contains: Tris-acetate, 20 raM, pH 7.2 ATP, 0.5 mM MgCI2, 10 mM UTP, 0.5 mM L-glutamine, 2 mM GTP, 0.1 mM Ammonia reaction mixture was similar except: (NH4)2 SO4, 10 mM, replaced L-glutamine and GTP, and the assay was carried out at pH 8.15 with Tris-acetate, 20 mM. Procedure. The substrates were preincubated for 3 min in cuvettes at 38°C. The enzyme preparation was added and contents mixed. The linear increase in absorbance at 291 nm was measured as a function of time on a Gilford recording spectrophotometer. Blank rates were obtained by omitting UTP from the reaction mixture. One unit of enzyme activity is defined as the amount of enzyme catalyzing the formation of 1 /zmole of CTP per minute in 1.0 ml of reaction mixture at 38°C. Purification Procedure All operations are performed at 0-4°C. The original purification procedure 4 was modified to give a homogeneous protein. 5-7
Crude Extract. To 850 g of E. coli B were added 1000 ml of 20 mM sodium phosphate buffer (pH 7.0) which contained 1 mM sodium EDTA and 2 mM L-glutamine. The cells were disrupted for 10 min in a 10-kc Raytheon sonic oscillator in 75-ml quantities. The cell debris was removed by centrifugation at 13,000 g for 45 min. The temperature was maintained below 6°C throughout subsequent steps. Streptomycin Sulfate Treatment. To 1650 ml of the above extract 800 ml of 10% streptomycin sulfate were added with stirring over a period of 2 hr. The resulting suspension was centrifuged at 13,000 g for 30 min to remove the precipitate. First Ammonium Sulfate Treatment. To 2300 ml of the above supernatant, 575 g of ammonium sulfate (enzyme grade) were added with stirring over a 60-rain period. The resultant precipitate was ree A. Levitzki and D. E. Koshland, Jr., Biochemistry 10, 3365 (1971). 7 A. Levitzki and D. E. Koshland, Jr., Biochim. Biophys. Acta 206, 473 (1970).
[12]
CYTIDINE TRIPHOSPHATE SYNTHETASE
81
moved by centrifugation at 13,000 g for 30 min and dissolved in 1000 ml of the original buffer.
Second Ammonium Sulfate Treatment. To 1050 ml of above solution, 200 g of ammonium sulfate were added, with stirring over a period of 45 min. The precipitate was removed by centrifugation at 13,000 g for 30 min. To the remaining supernatant, an additional 60 g of ammonium sulfate were added with stirring over a 20-min period. The precipitate was dissolved in 150 ml of the original buffer, and the 175 ml which resulted were eluted in five batches from a column of Sephadex G50 (6 × 40 cm) that had been previously equilibrated with 20 mM, sodium phosphate buffer, pH 7.4, 1 mM EDTA, 4 mM L-glutamine, and 70 mM fl-mercaptoethanol. This buffer was used in all subsequent steps. DEAE-Sephadex Chromatography. The desalted enzyme solution was applied to a DEAE-Sephadex A-50 column (6 × 30 cm) equilibrated with the above buffer, washed with 200 ml of the same buffer, and eluted with a linear gradient from 0-0.20 M (NH4)2SO4. The total gradient volume was 2400 ml, and 9-ml fractions were collected at a flow rate of 1 ml/min. The enzyme activity eluted midway through the gradient at 0.08-0.09 M (NH4)2SO4. The peak enzyme-containing fractions were pooled, precipitated with 55% saturated ammonium sulfate, and desalted by passage through a Sephadex G100 column (4 × 75 cm) at a flow rate of 60 ml/hr. Gel Filtration. The fractions from the Sephadex column with maximum activity were pooled and 3.0 ml were applied to a Biogel A 0.5 M column (2 × 85 cm) that had been equilibrated with buffer containing 0.75 M ATP, 0.75 M UTP, and 0.01 M MgClz. Under these conditions it was found 5'7 that the enzyme elutes as a tetramer of 210,000 molecular weight. The fractions containing maximum activity are pooled, precipitated with 55% ammonium sulfate, and reapplied to the Biogel A 0.5 M column in the absence of substrates. Under these conditions the activity elutes from the column at a point corresponding to a molecular weight of 105,000. A final purification is obtained by precipitating the pooled enzyme-containing fractions with 60% ammonium sulfate and applying 2.0 ml of the enzyme to a column of Sephadex G200 (2.5 × 90 cm) equilibrated with buffer without nucleotides. The enzyme eluting from the column is pure as judged from polyacrylamide gel electrophoresis. Comments on the Purification Procedure. The ammonium sulfate, DEAE-Sephadex A50, and gel filtration steps result in good purification with minimal loss of activity and good yields. The aggregation of the enzyme in the presence of substrate effectors provides a unique way to separate CTP synthetase from other proteins which copurify with it and
82
[12]
De Novo PYRIMIDINE BIOSYNTHESIS PURIFICATIONOF CYTIDINETRIPHOSPHATESYNTHETASE
Step
Activity (units)
Streptomycin sulfate Third (NH4hSO4 DEAE-Sephadex Sephadex G-100 Biogel A (0.5 M) + ATP + UTP + MgC12 Biogel A (0.5 M), no nucleotides Sephadex G-200
Yield of pooled Specificactivity fractions (/~molesCTP/min/mg) (%)
390 260 160 150 150
-0.05 0.30 1.30 2.00
112 100
4.60 5.80-6.10
100 67 41 38 38 29 26
otherwise would be difficult to remove. The overall purification from the crude extract is about 1000-fold and routinely resulted in a final yield of about 25%. A summary of the purification procedure is given in the table. P r o p e r t i e s o f the P u r i f i e d E n z y m e Requirements. The amination of U T P by the glutamine assay shows strict dependence on ATP, Mg 2+, and G T P with a p H optimum of 7.0 in Tris-acetate buffer. The ammonia assay does not require the allosteric effector G T P and has a p H optimum o f 8.2. The pure e n z y m e when stored at a concentration o f 1-4 mg/ml at 4°(2 in 20 mM sodium phosphate buffer, p H 7.2, 2 m M L-glutamine, l m M E D T A , 70 m M / 3 mercaptoethanol, and 20% glycerol is stable for 3 months. E n z y m e solutions that have lost activity may be reactivated by the addition of/3mercaptoethanol. Structure. CTP synthetase exists as a dimer in the absence o f substrates with a molecular weight o f 105,000 -- 2000 as measured by equilibrium centrifugation or gel filtration on Sephadex G200. 5 The e n z y m e can be split under denaturing conditions in guanidine hydrochloride or urea to two identical subunits with molecular weight o f 52,000. ~ Polyacrylamide gel electrophoresis of the purified protein at p H 8.0 and 9.5 in the presence o f 8 M urea gave a single band. s In the presence of U T P , Mg ~+, and A T P at concentrations used in the assay, the e n z y m e aggregates to a tetrameric form with molecular weight of 210,000 at s A. Levitzki, W. B. Stallcup, and D. E. Koshland, Jr., Biochemistry 10, 3371 (1971).
[12]
CYTIDINE TRIPHOSPHATE SYNTHETASE
83
protein concentrations as low as 5 /zg/ml. 5"9 The enzyme cannot be denatured to its monomeric form at pH values near its isoelectric point, 5.5. 8 CTP synthetase from mammalian liver has also been found to aggregate in the presence of ATP and UTP.I° Kinetics and Mechanism. Purified CTP synthetase exhibits positive cooperativity for the substrates ATP and UTP T M and negative cooperativity for an effector GTP 12"1a and the substrate L-glutamine. 5's'11 The initial kinetic analysis of the reaction indicated strong site-site interactions for ATP and UTP with Hill coefficients of 3.8 and 3.4, respectively. The high degree of cooperativity was in part explained by the substrate-induced dimer-tetramer aggregation. Both UTP and ATP were able to bring about this transition separately but were most efficient in combination. Initial studies 4 on the role of GTP indicated that this compound had no effect on the ammonia reaction but greatly accelerated the glutamine reaction. A detailed kinetic study TM provided evidence that saturating levels of GTP increased the Vmax of the glutamine reaction 10-fold and decreased the Km for glutamine by a factor of six. The CTP synthetase from mammalian liver has not been found to exhibit negative cooperativity for GTP binding.13 The effect of GTP on the formation of the enzyme-glutamine complex was verified by measuring the reaction of enzyme with the glutamine analog 6-diazo-5-oxonorleucine (DON). It was found that GTP accelerates the inactivation of CTP synthetase by enhancing the reaction of the affinity label DON with essential SH groups, s'a2 The interaction of CTP synthetase with DON initially presented an enigma since only two molecules were found to react per tetramer and to destroy activity. ~ This result was subsequently shown to be an extreme case of negative cooperativity where the DON-induced conformational changes are transmitted to remaining subunits and prevent binding of unfilled sites. 8 This phenomena was termed "half of the sites reactivity" and was found to occur in other proteins as well.
9 A. 10 R. 11 A. 12 A. 13 C.
Levitzki and D. E. Koshland, Jr., Biochemistry. 11, 247 (1972). P. McPartland and H. Weinfeld, J. Biol. Chem. 251, 4372 (1976). Levitzki and D. E. Koshland, Jr., Proc, Natl. Acad. Sci. U.S.A. 62, 1121 (1969). Levitzki and D. E. Koshland, Jr., Biochemistry 11, 241 (1972). R. Savage, Jr. and H. Weinfeld, J. Biol. Chem. 245, 2529 (1970).
CYTIDINE TRIPHOSPHATE SYNTHETASE
79
which is close to the value (7-8 pM) reported for the enzyme from brewer' s yeast. 15
Inhibitors. The enzyme is 70-100% inhibited by p-chloromercuribenzoate, 5,5'-dithiobis(2-nitrobenzoate), or Hg ~+ at 10 pAL but is not significantly inhibited by iodoacetate and iodoacetamide at 1 mM. GMP, GDP, CMP, and CDP are not much inhibitory at 0.5-1 mM. (Strong inhibitions observed previously 1 must have been due to some technical errors.) Uridine nucleotides and EDTA show no inhibition in the standard assay. 15 R. E. H a n d s c h u m a c h e r , J. Biol. Chem. 235, 2917 (1960).
[12] C y t i d i n e T r i p h o s p h a t e
Synthetase
By CEDRIC LONG and DANIEL E. KOSHLAND, JR. Cytidine triphosphate synthetase (CTPS) was initially characterized from Escherichia coli by Lieberman 1 and shown to catalyze the formation of CTP from UTP, ATP, magnesium, and ammonia. A similar reaction was later demonstrated in mammalian tissues 2 and bacteria 3 which used L-glutamine as an amino donor and required a guanosine nucleotide for maximum activities. Following these observations the enzyme was extensively purified from E. coli, and a kinetic analysis revealed a high degree of cooperativity in substrate binding to the enzyme. 4 Thus, both UTP and ATP showed strong site-site interactions, and GTP was found to be an allosteric effector for the glutamine reaction. Part of the high positive cooperativity was shown to proceed through conformational changes involving substrate-induced dimertetramer aggregation. 5 Such features have made CTPS an intriguing model for regulatory proteins. Assay Method
Principle. The enzyme assay measures the amination of UTP spectrophotometrically at 291 nm where UTP has an extinction coefficient I I. L i e b e r m a n , J. Biol. Chem. 222, 675 (1956). 2 H. O. K a m m e n and R. B. Hurlbert, Cancer Res. 19, 654 (1959). a K. B. Chakraborty and R. B. Hurlbert, Biochim. Biophys. Acta 4 7 , 6 0 7 (1961). 4 C. W. L o n g a n d A. B. Pardee, J. Biol. Chem. 242, 4715 (1967). C. W. Long, A. Levitzki, and D. E. K o s h l a n d , Jr., J. Biol. Chem. 245, 80 (1970).
M E T H O D S IN E N Z Y M O L O G Y , VOL. LI
Copyright © 1978by AcademicPress. Inc. All rights of reproduction in any form reserved. 1SBN 0-12-181951-5
80
De Novo
PYRIMID1NE BIOSYNTHESIS
[12]
of 182 and CTP 1520 at pH 7.0. The assay can be carried out with either L-glutamine or ammonia serving as the amino group donor.
Reagents L-glutamine reaction mixture (total of 1.0 ml) contains: Tris-acetate, 20 raM, pH 7.2 ATP, 0.5 mM MgCI2, 10 mM UTP, 0.5 mM L-glutamine, 2 mM GTP, 0.1 mM Ammonia reaction mixture was similar except: (NH4)2 SO4, 10 mM, replaced L-glutamine and GTP, and the assay was carried out at pH 8.15 with Tris-acetate, 20 mM. Procedure. The substrates were preincubated for 3 min in cuvettes at 38°C. The enzyme preparation was added and contents mixed. The linear increase in absorbance at 291 nm was measured as a function of time on a Gilford recording spectrophotometer. Blank rates were obtained by omitting UTP from the reaction mixture. One unit of enzyme activity is defined as the amount of enzyme catalyzing the formation of 1 /zmole of CTP per minute in 1.0 ml of reaction mixture at 38°C. Purification Procedure All operations are performed at 0-4°C. The original purification procedure 4 was modified to give a homogeneous protein. 5-7
Crude Extract. To 850 g of E. coli B were added 1000 ml of 20 mM sodium phosphate buffer (pH 7.0) which contained 1 mM sodium EDTA and 2 mM L-glutamine. The cells were disrupted for 10 min in a 10-kc Raytheon sonic oscillator in 75-ml quantities. The cell debris was removed by centrifugation at 13,000 g for 45 min. The temperature was maintained below 6°C throughout subsequent steps. Streptomycin Sulfate Treatment. To 1650 ml of the above extract 800 ml of 10% streptomycin sulfate were added with stirring over a period of 2 hr. The resulting suspension was centrifuged at 13,000 g for 30 min to remove the precipitate. First Ammonium Sulfate Treatment. To 2300 ml of the above supernatant, 575 g of ammonium sulfate (enzyme grade) were added with stirring over a 60-rain period. The resultant precipitate was ree A. Levitzki and D. E. Koshland, Jr., Biochemistry 10, 3365 (1971). 7 A. Levitzki and D. E. Koshland, Jr., Biochim. Biophys. Acta 206, 473 (1970).
[12]
CYTIDINE TRIPHOSPHATE SYNTHETASE
81
moved by centrifugation at 13,000 g for 30 min and dissolved in 1000 ml of the original buffer.
Second Ammonium Sulfate Treatment. To 1050 ml of above solution, 200 g of ammonium sulfate were added, with stirring over a period of 45 min. The precipitate was removed by centrifugation at 13,000 g for 30 min. To the remaining supernatant, an additional 60 g of ammonium sulfate were added with stirring over a 20-min period. The precipitate was dissolved in 150 ml of the original buffer, and the 175 ml which resulted were eluted in five batches from a column of Sephadex G50 (6 × 40 cm) that had been previously equilibrated with 20 mM, sodium phosphate buffer, pH 7.4, 1 mM EDTA, 4 mM L-glutamine, and 70 mM fl-mercaptoethanol. This buffer was used in all subsequent steps. DEAE-Sephadex Chromatography. The desalted enzyme solution was applied to a DEAE-Sephadex A-50 column (6 × 30 cm) equilibrated with the above buffer, washed with 200 ml of the same buffer, and eluted with a linear gradient from 0-0.20 M (NH4)2SO4. The total gradient volume was 2400 ml, and 9-ml fractions were collected at a flow rate of 1 ml/min. The enzyme activity eluted midway through the gradient at 0.08-0.09 M (NH4)2SO4. The peak enzyme-containing fractions were pooled, precipitated with 55% saturated ammonium sulfate, and desalted by passage through a Sephadex G100 column (4 × 75 cm) at a flow rate of 60 ml/hr. Gel Filtration. The fractions from the Sephadex column with maximum activity were pooled and 3.0 ml were applied to a Biogel A 0.5 M column (2 × 85 cm) that had been equilibrated with buffer containing 0.75 M ATP, 0.75 M UTP, and 0.01 M MgClz. Under these conditions it was found 5'7 that the enzyme elutes as a tetramer of 210,000 molecular weight. The fractions containing maximum activity are pooled, precipitated with 55% ammonium sulfate, and reapplied to the Biogel A 0.5 M column in the absence of substrates. Under these conditions the activity elutes from the column at a point corresponding to a molecular weight of 105,000. A final purification is obtained by precipitating the pooled enzyme-containing fractions with 60% ammonium sulfate and applying 2.0 ml of the enzyme to a column of Sephadex G200 (2.5 × 90 cm) equilibrated with buffer without nucleotides. The enzyme eluting from the column is pure as judged from polyacrylamide gel electrophoresis. Comments on the Purification Procedure. The ammonium sulfate, DEAE-Sephadex A50, and gel filtration steps result in good purification with minimal loss of activity and good yields. The aggregation of the enzyme in the presence of substrate effectors provides a unique way to separate CTP synthetase from other proteins which copurify with it and
82
[12]
De Novo PYRIMIDINE BIOSYNTHESIS PURIFICATIONOF CYTIDINETRIPHOSPHATESYNTHETASE
Step
Activity (units)
Streptomycin sulfate Third (NH4hSO4 DEAE-Sephadex Sephadex G-100 Biogel A (0.5 M) + ATP + UTP + MgC12 Biogel A (0.5 M), no nucleotides Sephadex G-200
Yield of pooled Specificactivity fractions (/~molesCTP/min/mg) (%)
390 260 160 150 150
-0.05 0.30 1.30 2.00
112 100
4.60 5.80-6.10
100 67 41 38 38 29 26
otherwise would be difficult to remove. The overall purification from the crude extract is about 1000-fold and routinely resulted in a final yield of about 25%. A summary of the purification procedure is given in the table. P r o p e r t i e s o f the P u r i f i e d E n z y m e Requirements. The amination of U T P by the glutamine assay shows strict dependence on ATP, Mg 2+, and G T P with a p H optimum of 7.0 in Tris-acetate buffer. The ammonia assay does not require the allosteric effector G T P and has a p H optimum o f 8.2. The pure e n z y m e when stored at a concentration o f 1-4 mg/ml at 4°(2 in 20 mM sodium phosphate buffer, p H 7.2, 2 m M L-glutamine, l m M E D T A , 70 m M / 3 mercaptoethanol, and 20% glycerol is stable for 3 months. E n z y m e solutions that have lost activity may be reactivated by the addition of/3mercaptoethanol. Structure. CTP synthetase exists as a dimer in the absence o f substrates with a molecular weight o f 105,000 -- 2000 as measured by equilibrium centrifugation or gel filtration on Sephadex G200. 5 The e n z y m e can be split under denaturing conditions in guanidine hydrochloride or urea to two identical subunits with molecular weight o f 52,000. ~ Polyacrylamide gel electrophoresis of the purified protein at p H 8.0 and 9.5 in the presence o f 8 M urea gave a single band. s In the presence of U T P , Mg ~+, and A T P at concentrations used in the assay, the e n z y m e aggregates to a tetrameric form with molecular weight of 210,000 at s A. Levitzki, W. B. Stallcup, and D. E. Koshland, Jr., Biochemistry 10, 3371 (1971).
[12]
CYTIDINE TRIPHOSPHATE SYNTHETASE
83
protein concentrations as low as 5 /zg/ml. 5"9 The enzyme cannot be denatured to its monomeric form at pH values near its isoelectric point, 5.5. 8 CTP synthetase from mammalian liver has also been found to aggregate in the presence of ATP and UTP.I° Kinetics and Mechanism. Purified CTP synthetase exhibits positive cooperativity for the substrates ATP and UTP T M and negative cooperativity for an effector GTP 12"1a and the substrate L-glutamine. 5's'11 The initial kinetic analysis of the reaction indicated strong site-site interactions for ATP and UTP with Hill coefficients of 3.8 and 3.4, respectively. The high degree of cooperativity was in part explained by the substrate-induced dimer-tetramer aggregation. Both UTP and ATP were able to bring about this transition separately but were most efficient in combination. Initial studies 4 on the role of GTP indicated that this compound had no effect on the ammonia reaction but greatly accelerated the glutamine reaction. A detailed kinetic study TM provided evidence that saturating levels of GTP increased the Vmax of the glutamine reaction 10-fold and decreased the Km for glutamine by a factor of six. The CTP synthetase from mammalian liver has not been found to exhibit negative cooperativity for GTP binding.13 The effect of GTP on the formation of the enzyme-glutamine complex was verified by measuring the reaction of enzyme with the glutamine analog 6-diazo-5-oxonorleucine (DON). It was found that GTP accelerates the inactivation of CTP synthetase by enhancing the reaction of the affinity label DON with essential SH groups, s'a2 The interaction of CTP synthetase with DON initially presented an enigma since only two molecules were found to react per tetramer and to destroy activity. ~ This result was subsequently shown to be an extreme case of negative cooperativity where the DON-induced conformational changes are transmitted to remaining subunits and prevent binding of unfilled sites. 8 This phenomena was termed "half of the sites reactivity" and was found to occur in other proteins as well.
9 A. 10 R. 11 A. 12 A. 13 C.
Levitzki and D. E. Koshland, Jr., Biochemistry. 11, 247 (1972). P. McPartland and H. Weinfeld, J. Biol. Chem. 251, 4372 (1976). Levitzki and D. E. Koshland, Jr., Proc, Natl. Acad. Sci. U.S.A. 62, 1121 (1969). Levitzki and D. E. Koshland, Jr., Biochemistry 11, 241 (1972). R. Savage, Jr. and H. Weinfeld, J. Biol. Chem. 245, 2529 (1970).