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[31b] 2-Ketogluconokinase from Aerobacter aerogenes
[3 lb]
2-KETOGLUCONOKINASE
291
observed. All purified preparations except one displayed the spectrum for reduced eytochrome c on addition of gluconate or hydrosulfite. Spectral evidence and activation of apo-D-amino acid oxidase by extracts of boiled enzyme preparations suggest the presence of fiavin or FAD, but the presence and function of these components need further proof. Metal ions are not required. In other purified preparations obtained without acetone drying, bacterial cytochromes were reduced by gluconate. 2Ketogluconate has been identified as the reaction product.
[ 31b]
2-Ketogluconokinase from Aerobacter
aerogenes
2-Ketogluconate q- ATP -~ 2-Keto-6-phosphoglueo~ate q- ADP B y W. A. WOOD and E. W. F~MPTON
Assay Method Principle. The general manometric assay for kinases of Colowick and Kalckar 1 which makes use of the new acidic group formed in transphosphorylation to evolve C02 from bicarbonate buffer-~ is employed. Alternate assays are based on (1) the loss of semicarbazide-reacting material s (2-ketogluconate, 30-minute heating) after precipitation of the phosphate esters with Ba(OH)2-ZnS04, 4 or (2) the oxidation of T P N H or DPNH in the presence of excess 2-keto-6-phosphogluconate reductase2 Reagents
0.1 M MgC12. 0.28 M NaHCO~. 0.1M Na 2-ketogluconate. Calcium 2-ketogluconate "2H~O was converted to the sodium salt by passage through an IR-120 (H ÷) column followed by neutralization of the 2-ketoglueonic acid to pH 7.5 with 2.5 N NaOH. 0.1 M Na~. ATP "2H20 (Pabst), crystalline, pH 7.5. Procedure. To the main compartment of a 5-ml. Warburg flask equipped with venting cock were added 0.2 ml. of MgCI~, 0.1 ml. of NaHC0~, 2-ketogluconokinase, and water to 0.85 ml. To the side arm of the flask were added 0.15 ml. of Na 2-ketoglueonateand 0.2 ml. of ATP.
1S. P. Colowick and H. M. Kalckar, J. Biol. Chem. 148, 117 (1943). S. P. Colowick, "The Enzymes," Vol. II, pp. 114--150, 1951. 3j. MacGee and M. Doudoroff,J. Biol. Chem. 210, 617 (1954). 4M. Somogyi,J. Biol. Chem. 160, 69 (1945). See Vol. V [31c].
292
ENZYMES OF CARBOHYDRATE METABOLISM
[31b]
The flasks were then placed in the water bath, gassed with N2-C02 ( 9 5 % - 5 % ) for 10 minutes, and the cocks closed. After 5 minutes of further equilibration, the contents of the side arm was tipped, and the evolution of CO_. was recorded between 5 and 25 minutes after tipping. An A T P a s e control (without substrate) was run for each fraction tested. Definition o] Unit. One unit of enzyme is defined as the amount that causes the phosphorylation of 1 micromole of 2-ketogluconate per hour. This rate is calculated from the rate of C02 evolution between the fifth and twenty-fifth minutes after tipping and after subtracting the amount of C02 evolved due to ATPase activity. Thus 1 unit is equivalent to the net evolution of 22.4 ~l. of CO-" per hour. Specific activity is expressed as units per milligrams of protein, where protein is determined by the method of Lowry et al2 with crystalline bovine serum albumin as a standard. Application of the Assay M e t h o d to Crude Preparations. Approximately equal amounts of ATPase and 2-ketogluconokinase are present in crude extracts of A. aerogenes and P. fluorescens. 7 The kinase also has been demonstrated by this method in A. cloacae s and has been purified from L. mesenteroides2
Purification Procedure Step 1. Growth o] Culture and Preparation o] Crude Extract. Although 2-ketogluconokinase is induced and is functional in glucose metabolism by P. fluorescens, 1°-12 a higher concentration of kinase is present in 2-ketogluconate-grown A. aerogenes, strain P R L - R 3 . This organism was grown in the mineral medium of Davis and Mingioli, 1~ which was modified to the extent t h a t N a 2-ketogluconate was substituted for glucose. F o r preparation of large quantities of cells, 14 1. of medium in a 20-1. carboy was inoculated with 1 1. of culture grown in the same medium in Fernbaeh flasks for 18 to 24 hours at 35% The carboy was fitted with stirring and aeration devices (1725 r.p.m., 2 vol. of air per minute) to obtain maximum aeration. The culture was harvested after 5 hours, washed once with 0.85% NaC1, and suspended in 500 ml.
cO. H. Lowry, N. J. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). TS. A. Narrod and W. A. Wood, J. Biol. Chem. 220, 45 (1956). sj. DeLey, Enzymologia 17, 55 (1954). *O. Ciferri, E. R. Blakely, and F. J. Simpson, Can. J. Microblol. 5, 277 (1959). '°W. A. Wood and R. F. Schwerdt, J. Biol. Chem. 201, 501 (1953). "W. A. Wood, S. A. Narrod, and B. C. Hertlein, Abstr. Communs. 3rd Intern. Congr. Biochem., Brussels p. 55 (1959). E. W. Frampton and W. A. Wood, BacterioI. Proc. p. 122 (1957). 'SB. D. Davis and E. S. Mingioli, J. Bacteriol. 6@, 17 (1950).
[31b]
2-KETOGLUCONOKINASE
293
of water. Extracts were prepared by sonic oscillation at 10 kc. for 10 minutes followed by c~ntrifugation at 7000 X g for 30 minutes. All subsequent operations were performed at 0 ° to 5 °. Precipitates were separated by centrifugation at 15,000 X g for 10 minutes and dissolved in l0 -3 M sodium ethylenediaminetetraacetate (EDTA), pH 7.5. Step 2. Removal o] Nucleic Acids. To a cell-free "extract (420 ml., 16.8 mg. of protein per milliliter14), solid ammonium sulfate was added to 0.1 M concentration. Two per cent protamine sulfate, pH 5, was added with stirring to the extent of 20% of the extract volume. The precipitate was removed by centrifugation and discarded. The supernatant solution, 500 ml., contained 7.7 mg. of protein per milliliter and had a 280/260-m~ ratio of 0.89. Step 3. Ammonium Sul]ate Fractionation. Solid ammonium sulfate was added to 50% saturation, 1~ and the precipitate was removed by centrifugation and discarded. The supernatant solution was then saturated with solid ammonium sulfate and centrifuged. The precipitate was dissolved in 100 ml. of EDTA (AmS04 I, 100 ml., 17.4 mg. of protein per milliliter). Step ~. Ammonium Sul]ate Fractionation. Solid ammonium sulfate was added to AmSO~ I in the same manner to 40% of saturation, and the precipitate obtained was discarded. Ammonium sulfate was added to the supernatant solution to 50% of saturation, and the recovered precipitate was dissolved in 50 ml. of EDTA buffer (AmSO, II, 50 ml., 5.2 mg. of protein per milliliter). A portion of the 2-ketogluconokinase precipitated between 50% and 60% ammonium sulfate saturation, but was discarded because of its lower specific activity. Step 5. Calcium Phosphate Gel Adsorption and Elution. AmSO~ II was diluted with an equal volume of 0.05 M ammonium sulfate, and the pH was adjusted to 5.5 with 1 N acetic acid. The protein content was 2.6 mg./ml., and the ammonium sulfate concentration was 0.047 M. Calcium phosphate gel (7.2 mg. dry weight per milliliter) was added to the stirring solution by syringe. After standing for 5 minutes the gel was recovered by centrifugation and washed with 50 ml. of water and with 50 ml. of 0.005 M KH2PQ, pH 5.5. The kinase was eluted by stirring the gel for 5 minutes with two 50-ml. portions of 0.0075 M KH~P04, pH 7.5, and two portions of 0.01 M KH2P04, pH 7.5. The elutes were combined (200 ml., 0.33 mg. of protein per milligram). Step 6. Ammonium Sul]ate Fractionation. Solid ammonium sulfate was added to obtain a precipitate between 50% and 75% of saturation. '~It has been found subsequently that better results are obtained in the protamine treatment if the protein concentration is less than 10 mg./ml. ,5A. A. Green and W. L. Hughes, see Vol. I [10] p. 67.
294
ENZYMES OF CARBOHYDRATE METABOLISM
[31b]
The precipitate was dissolved in E D T A buffer ( a m m o n i u m sulfate I I I , 20 ml., 2.08 mg. of protein per milliliter). A s u m m a r y of the purification procedure is given in the table. SUMMARY OF PURIFICATION PROCEDURE
Step
Crude extract Protamine supernatant AmS04 I (0.5-1.0 saturation) AreS04 II (0.4-0.5 saturation) Calcium phosphate gel eluates AreS04 III (0.5-0.75 saturation)
Specific Total Recovery, activity, activity, % units/mg, units protein
Purification, -fold
25,000 22,600 23,400 20,500 (25,655) 20,180
-1.6 6.15 20.2 87.2 124
100 90.5 93.6 82.0 -80.6
4 6 24 79 341 485
Properties F o r t y per cent of the activity of the purified enzyme was lost during storage at - - 1 4 ° for 14 months. The kinase was free of gluconokinase, 2-keto-6-phosphogluconate reductase, and A T P a s e activities. T h e reaction velocity increased up to p H 8, which is the upper limit of the HC08-CO_~ buffer system. 1G The purified fraction displayed an absolute requirement for m a g nesium ions but was not stimulated by glutathione and E D T A as had been found for L-ribulokinase derived from the same organism. 17 The K,~ for 2-ketogluconate is 1.5 X 10 -5 M. T h e preparation was used to prepare 2-keto-6-phosphogluconate in quantity. ~ w. w. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques and Related Methods of the Study of Tissue Metabolism." Burgess Publishing Co., Minneapolis, 1941. 1; F. J. Simpson and W. A. Wood, J. Biol. Chem. 230, 473 (1958).
2-KETOGLUCONOKINASE
291
observed. All purified preparations except one displayed the spectrum for reduced eytochrome c on addition of gluconate or hydrosulfite. Spectral evidence and activation of apo-D-amino acid oxidase by extracts of boiled enzyme preparations suggest the presence of fiavin or FAD, but the presence and function of these components need further proof. Metal ions are not required. In other purified preparations obtained without acetone drying, bacterial cytochromes were reduced by gluconate. 2Ketogluconate has been identified as the reaction product.
[ 31b]
2-Ketogluconokinase from Aerobacter
aerogenes
2-Ketogluconate q- ATP -~ 2-Keto-6-phosphoglueo~ate q- ADP B y W. A. WOOD and E. W. F~MPTON
Assay Method Principle. The general manometric assay for kinases of Colowick and Kalckar 1 which makes use of the new acidic group formed in transphosphorylation to evolve C02 from bicarbonate buffer-~ is employed. Alternate assays are based on (1) the loss of semicarbazide-reacting material s (2-ketogluconate, 30-minute heating) after precipitation of the phosphate esters with Ba(OH)2-ZnS04, 4 or (2) the oxidation of T P N H or DPNH in the presence of excess 2-keto-6-phosphogluconate reductase2 Reagents
0.1 M MgC12. 0.28 M NaHCO~. 0.1M Na 2-ketogluconate. Calcium 2-ketogluconate "2H~O was converted to the sodium salt by passage through an IR-120 (H ÷) column followed by neutralization of the 2-ketoglueonic acid to pH 7.5 with 2.5 N NaOH. 0.1 M Na~. ATP "2H20 (Pabst), crystalline, pH 7.5. Procedure. To the main compartment of a 5-ml. Warburg flask equipped with venting cock were added 0.2 ml. of MgCI~, 0.1 ml. of NaHC0~, 2-ketogluconokinase, and water to 0.85 ml. To the side arm of the flask were added 0.15 ml. of Na 2-ketoglueonateand 0.2 ml. of ATP.
1S. P. Colowick and H. M. Kalckar, J. Biol. Chem. 148, 117 (1943). S. P. Colowick, "The Enzymes," Vol. II, pp. 114--150, 1951. 3j. MacGee and M. Doudoroff,J. Biol. Chem. 210, 617 (1954). 4M. Somogyi,J. Biol. Chem. 160, 69 (1945). See Vol. V [31c].
292
ENZYMES OF CARBOHYDRATE METABOLISM
[31b]
The flasks were then placed in the water bath, gassed with N2-C02 ( 9 5 % - 5 % ) for 10 minutes, and the cocks closed. After 5 minutes of further equilibration, the contents of the side arm was tipped, and the evolution of CO_. was recorded between 5 and 25 minutes after tipping. An A T P a s e control (without substrate) was run for each fraction tested. Definition o] Unit. One unit of enzyme is defined as the amount that causes the phosphorylation of 1 micromole of 2-ketogluconate per hour. This rate is calculated from the rate of C02 evolution between the fifth and twenty-fifth minutes after tipping and after subtracting the amount of C02 evolved due to ATPase activity. Thus 1 unit is equivalent to the net evolution of 22.4 ~l. of CO-" per hour. Specific activity is expressed as units per milligrams of protein, where protein is determined by the method of Lowry et al2 with crystalline bovine serum albumin as a standard. Application of the Assay M e t h o d to Crude Preparations. Approximately equal amounts of ATPase and 2-ketogluconokinase are present in crude extracts of A. aerogenes and P. fluorescens. 7 The kinase also has been demonstrated by this method in A. cloacae s and has been purified from L. mesenteroides2
Purification Procedure Step 1. Growth o] Culture and Preparation o] Crude Extract. Although 2-ketogluconokinase is induced and is functional in glucose metabolism by P. fluorescens, 1°-12 a higher concentration of kinase is present in 2-ketogluconate-grown A. aerogenes, strain P R L - R 3 . This organism was grown in the mineral medium of Davis and Mingioli, 1~ which was modified to the extent t h a t N a 2-ketogluconate was substituted for glucose. F o r preparation of large quantities of cells, 14 1. of medium in a 20-1. carboy was inoculated with 1 1. of culture grown in the same medium in Fernbaeh flasks for 18 to 24 hours at 35% The carboy was fitted with stirring and aeration devices (1725 r.p.m., 2 vol. of air per minute) to obtain maximum aeration. The culture was harvested after 5 hours, washed once with 0.85% NaC1, and suspended in 500 ml.
cO. H. Lowry, N. J. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). TS. A. Narrod and W. A. Wood, J. Biol. Chem. 220, 45 (1956). sj. DeLey, Enzymologia 17, 55 (1954). *O. Ciferri, E. R. Blakely, and F. J. Simpson, Can. J. Microblol. 5, 277 (1959). '°W. A. Wood and R. F. Schwerdt, J. Biol. Chem. 201, 501 (1953). "W. A. Wood, S. A. Narrod, and B. C. Hertlein, Abstr. Communs. 3rd Intern. Congr. Biochem., Brussels p. 55 (1959). E. W. Frampton and W. A. Wood, BacterioI. Proc. p. 122 (1957). 'SB. D. Davis and E. S. Mingioli, J. Bacteriol. 6@, 17 (1950).
[31b]
2-KETOGLUCONOKINASE
293
of water. Extracts were prepared by sonic oscillation at 10 kc. for 10 minutes followed by c~ntrifugation at 7000 X g for 30 minutes. All subsequent operations were performed at 0 ° to 5 °. Precipitates were separated by centrifugation at 15,000 X g for 10 minutes and dissolved in l0 -3 M sodium ethylenediaminetetraacetate (EDTA), pH 7.5. Step 2. Removal o] Nucleic Acids. To a cell-free "extract (420 ml., 16.8 mg. of protein per milliliter14), solid ammonium sulfate was added to 0.1 M concentration. Two per cent protamine sulfate, pH 5, was added with stirring to the extent of 20% of the extract volume. The precipitate was removed by centrifugation and discarded. The supernatant solution, 500 ml., contained 7.7 mg. of protein per milliliter and had a 280/260-m~ ratio of 0.89. Step 3. Ammonium Sul]ate Fractionation. Solid ammonium sulfate was added to 50% saturation, 1~ and the precipitate was removed by centrifugation and discarded. The supernatant solution was then saturated with solid ammonium sulfate and centrifuged. The precipitate was dissolved in 100 ml. of EDTA (AmS04 I, 100 ml., 17.4 mg. of protein per milliliter). Step ~. Ammonium Sul]ate Fractionation. Solid ammonium sulfate was added to AmSO~ I in the same manner to 40% of saturation, and the precipitate obtained was discarded. Ammonium sulfate was added to the supernatant solution to 50% of saturation, and the recovered precipitate was dissolved in 50 ml. of EDTA buffer (AmSO, II, 50 ml., 5.2 mg. of protein per milliliter). A portion of the 2-ketogluconokinase precipitated between 50% and 60% ammonium sulfate saturation, but was discarded because of its lower specific activity. Step 5. Calcium Phosphate Gel Adsorption and Elution. AmSO~ II was diluted with an equal volume of 0.05 M ammonium sulfate, and the pH was adjusted to 5.5 with 1 N acetic acid. The protein content was 2.6 mg./ml., and the ammonium sulfate concentration was 0.047 M. Calcium phosphate gel (7.2 mg. dry weight per milliliter) was added to the stirring solution by syringe. After standing for 5 minutes the gel was recovered by centrifugation and washed with 50 ml. of water and with 50 ml. of 0.005 M KH2PQ, pH 5.5. The kinase was eluted by stirring the gel for 5 minutes with two 50-ml. portions of 0.0075 M KH~P04, pH 7.5, and two portions of 0.01 M KH2P04, pH 7.5. The elutes were combined (200 ml., 0.33 mg. of protein per milligram). Step 6. Ammonium Sul]ate Fractionation. Solid ammonium sulfate was added to obtain a precipitate between 50% and 75% of saturation. '~It has been found subsequently that better results are obtained in the protamine treatment if the protein concentration is less than 10 mg./ml. ,5A. A. Green and W. L. Hughes, see Vol. I [10] p. 67.
294
ENZYMES OF CARBOHYDRATE METABOLISM
[31b]
The precipitate was dissolved in E D T A buffer ( a m m o n i u m sulfate I I I , 20 ml., 2.08 mg. of protein per milliliter). A s u m m a r y of the purification procedure is given in the table. SUMMARY OF PURIFICATION PROCEDURE
Step
Crude extract Protamine supernatant AmS04 I (0.5-1.0 saturation) AreS04 II (0.4-0.5 saturation) Calcium phosphate gel eluates AreS04 III (0.5-0.75 saturation)
Specific Total Recovery, activity, activity, % units/mg, units protein
Purification, -fold
25,000 22,600 23,400 20,500 (25,655) 20,180
-1.6 6.15 20.2 87.2 124
100 90.5 93.6 82.0 -80.6
4 6 24 79 341 485
Properties F o r t y per cent of the activity of the purified enzyme was lost during storage at - - 1 4 ° for 14 months. The kinase was free of gluconokinase, 2-keto-6-phosphogluconate reductase, and A T P a s e activities. T h e reaction velocity increased up to p H 8, which is the upper limit of the HC08-CO_~ buffer system. 1G The purified fraction displayed an absolute requirement for m a g nesium ions but was not stimulated by glutathione and E D T A as had been found for L-ribulokinase derived from the same organism. 17 The K,~ for 2-ketogluconate is 1.5 X 10 -5 M. T h e preparation was used to prepare 2-keto-6-phosphogluconate in quantity. ~ w. w. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques and Related Methods of the Study of Tissue Metabolism." Burgess Publishing Co., Minneapolis, 1941. 1; F. J. Simpson and W. A. Wood, J. Biol. Chem. 230, 473 (1958).