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[44] Preparation of 3-enolpyruvylshikimate 5-phosphate
360
[44]
AROMATIC AMINO ACIDS
[44] Preparation o f 3 - E n o l p y r u v y l s h i k i m a t e 5-Phosphate 1
By P. F. KNOWLES,J. G. LEVlN, and D. B. SPRINSON COOH
COOH
c s, + H2C= C--OPOs2-
71 : PO~- OH
OH
Shikimate 5- phosphate
Enolpyruvate phosphate
_-'-OsPO/
+ H2 "~"" "O--C--COOS OH
3-Enolpyruvyl shikimate 5-phosphate
HPO~-
Orthophosphate
Partially purified fractions of Escherichia coli are unable to convert 3-enolpyruvylshikimate 5-phosphate (ES-5-P) to chorismate unless special reducing conditions are provided, TM and are therefore suitable for the preparation of ES-5-P from shikimate-5-P and enolpyruvate-P. In the procedure described below an ammonium sulfate fraction of E. coli KI2 mutant 58-278 having a specific activity of 8 micromoles of ES-5-P per milligram of protein per hour 2 is used to prepare this compound. Similar results were obtained routinely with an ammonium sulfate fraction from E. coli B-37. TM This strain was used since it was available in large quantity in this laboratory. However, wild-type cells should be equally capable of serving as a source of enzyme, s A solution (475 ml) containing 0.5 millimole of potassium enolpyruvate-P, 4 0.5 millimole of potassium shikimate-5-P, e 5 millimoles of KF 13-Enolpyruvylshikimate 5-phosphate has also been prepared for use without purification by incubation of shikimate with a multienzyme complex from Neurospora which lacks chorismate synthase. (See article 48, footnote 8) aaH. Morell, M.J. Clark, P. F. Knowles, and D. B. Sprinson,J. Biol. Chem. 242, 82 (1967). 2j. G. Levin and D. B. Sprinson,J. Biol. Chem. 239, 1142 (1964). SFor assay of ES-5-P synthetase activity see E. Gollub, H. Zalkin, and D. B. Sprinson, J. Biol. Chem. 242, 5323 (1967). 4V. M. Clark and A. J. Kirby, Biochim. Biophys. Acta 78, 732 (1963); Biochem. Prep. 11, 101 (1966). A solution of 350 mg monocyclohexylammonium enolpyruvate-P in 4 ml of water is mixed vigorously for 2 minutes with 2 ml of washed Dowex-50 (H+). The resin is removed by centrifugation and washed twice with 1.5 ml portions of water. The combined supernatant solutions are adjusted immediately to pH 6.8-7.0 with i N KOH, assayed by the method of Kornberg and Pricer, 5 and made up to 0.1 M. 5A. Kornberg and W. E. Pricer, Jr.,J. Biol. Chem. 193,481 (1951). sp. F. Knowles and D. B. Sprinson, this volume [43a].
[44]
PREPARATION OF ES-5-P
361
(to inhibit phosphatase activity), and 25 millimoles of Tris-chloride buffer, pH 7.2, is incubated at 37 ° for 30 minutes. The 0.4 to 0.6 saturated (NH4)2SO4fraction prepared from E. coli KI2 58-278 (25 ml, 20 mg of protein per ml) ~ is added, and incubation is continued for 1 hour. The reaction mixture is brought to pH 8 by the addition of 1 N NaOH, heated for 5 minutes in a boiling water bath, and clarified by filtration. Assay of a sample (0.5 ml) for enoipyruvate-P 5 indicates the disappearance of approximately 80 % of this substrate.~ Dowex l-X8 (chloride form, 200-400 mesh) is allowed to stand overnight in 3 N HCI, washed until the washings are free of chloride, and thoroughly freed of fine particles. A column of resin is prepared 50 cm high and 3 cm in diameter, covered with a 0.5 cm layer of acid-washed (3 N HCI) Berkshire sand, and equilibrated with 0.01 M Tris-chloride buffer, pH 9.0 (by passagq of 3 liters of buffer overnight). The filtered incubation mixture is loaded on the column at a rate of 1.5 ml/minute, and the column is washed with 500 ml of 0.01 M Tris-chloride buffer, pH 9.0. Separation is effected by gradient elution at a rate of 1.5 ml/minute with a 5-liter reservoir of 0.6 M LiCI in 0.01 M Tris-chloride buffer, pH 9, and a constant volume mixing chamber containing 3 liters of the buffer without LiCI. Fractions (250, 15 ml each) are collected on a fraction collector and monitored automatically in an ultraviolet analyzer by measuring absorption at 240 m/z; the first 1000 ml of eluate may be collected in one batch. In a typical pattern, shikimate-5-P is found in fractions 90-100; enolpyruvylshikimate, in fractions 103-110; enolpyruvate-P, in fractions 114-125; and ES-5-P, in fractions 162-188. 2 The last three compounds are identified by heating in 0.1 N HCI in a boiling water bath for 10 minutes, and determining the resulting pyruvate as the 2,4-dinitrophenylhydrazone. s The combined fractions containing ES-5-P (350 micromoles) are concentrated to 50 ml by rotary evaporation under reduced pressure (bath temperature, 35°), and treated with 40 ml of 0.1 M barium acetate (6 times the theoretical quantity) and 270 ml of cold absolute ethanol. After standing overnight at 0°, the precipitate is removed by centrifugation, washed four times with 10 ml portions of 67% ethanol, and dried in a vacuum over P205. The yield is 810 mg~ containing 320 micromoles of the barium salt of ES-5-P. The crude product is stirred with 10 ml of water, the suspension is cleared by centrifugation, and the supernatant solution is removed with a pipette. The process of extraction is repeated seven more times. The extracts (approximately 80 ml) are combined, treated with 160 ml rThe yield of ES-5-P is only 50 to 60 % since some enolpyruvate-P is lost in side reactions. ST. E. Friedemann and G. E. Haugen,J. Biol. Chem. 147, 41.5 ( ! 94:3).
362
AROMATIC AMINO ACIDS
[45]
of absolute ethanol, and stored overnight at 0 °. The precipitate is collected by centrifugation, washed four times with 10 ml portions of cold 67 % ethanol, and dried in a vacuum over P205. The yield is 196 mg of white solid, containing 290 micromoles of the barium salt of ES-5-P. At 100 ° under reduced pressure, this salt shows a loss in weight of 5.4%; the theoretical loss for Ba2(ES-5-P) • 2 H 2 0 is 5.7%. On the assumption of a molecular weight of 630 for the dihydrate, the purity of the barium salt is 94%. This degree of purity is also indicated by an analysis for constituent groups. 2 The barium salt of ES-5-P (1.3 rag, 2.06 micromoles) is converted to the potassium salt. Shikimate (2.0 micromoles) is produced only after treatment with alkaline phosphatase followed by hydrolysis with dilute acid, indicating the absence of enolpyruvylshikimate and shikimate-5-P. Hydrolysis with dilute acid released 1.9 micromoles of pyruvate, and treatment with alkaline phosphatase released 2.0 micromoles of orthophosphate, which was otherwise absent. Assay for enolpyruvate-P 5 gave a negative result.
[45] Preparation of Chorismic Acid By F. GIBSON COOH
O---C OH
COOH
Chorismic acid
Chorismic acid [a-(5-carboxy-l,2-dihydro-2-hydroxyphenoxy)acrylic acid] has not been synthesized chemically, but is readily accumulated by cell suspensions of a multiply-blocked strain ofAerobacter aerogenes (62-1) with metabolic blocks in the biosynthetic pathways leading to tryptophan, phenylalanine, and tyrosine.' Because the organism used is capable of forming anthranilate from chorismate, the cells are grown in a medium containing tryptophan to repress anthranilate synthetase. 2 The cells are then transferred to an accumulation mixture from which the free acid is isolated. 2,a IM. I. Gibson a n d F. Gibson, Biochem..], 90, 248 (1964); see also Biochem. Prepn. 12, 94
(1968). 2F. (;ibson, Biochem..]. 90, 256 (1964). % M. Edwards a n d I J. M. J a c k m a n , Australian.]. (;hem. 18, 1227 (1965).
[44]
AROMATIC AMINO ACIDS
[44] Preparation o f 3 - E n o l p y r u v y l s h i k i m a t e 5-Phosphate 1
By P. F. KNOWLES,J. G. LEVlN, and D. B. SPRINSON COOH
COOH
c s, + H2C= C--OPOs2-
71 : PO~- OH
OH
Shikimate 5- phosphate
Enolpyruvate phosphate
_-'-OsPO/
+ H2 "~"" "O--C--COOS OH
3-Enolpyruvyl shikimate 5-phosphate
HPO~-
Orthophosphate
Partially purified fractions of Escherichia coli are unable to convert 3-enolpyruvylshikimate 5-phosphate (ES-5-P) to chorismate unless special reducing conditions are provided, TM and are therefore suitable for the preparation of ES-5-P from shikimate-5-P and enolpyruvate-P. In the procedure described below an ammonium sulfate fraction of E. coli KI2 mutant 58-278 having a specific activity of 8 micromoles of ES-5-P per milligram of protein per hour 2 is used to prepare this compound. Similar results were obtained routinely with an ammonium sulfate fraction from E. coli B-37. TM This strain was used since it was available in large quantity in this laboratory. However, wild-type cells should be equally capable of serving as a source of enzyme, s A solution (475 ml) containing 0.5 millimole of potassium enolpyruvate-P, 4 0.5 millimole of potassium shikimate-5-P, e 5 millimoles of KF 13-Enolpyruvylshikimate 5-phosphate has also been prepared for use without purification by incubation of shikimate with a multienzyme complex from Neurospora which lacks chorismate synthase. (See article 48, footnote 8) aaH. Morell, M.J. Clark, P. F. Knowles, and D. B. Sprinson,J. Biol. Chem. 242, 82 (1967). 2j. G. Levin and D. B. Sprinson,J. Biol. Chem. 239, 1142 (1964). SFor assay of ES-5-P synthetase activity see E. Gollub, H. Zalkin, and D. B. Sprinson, J. Biol. Chem. 242, 5323 (1967). 4V. M. Clark and A. J. Kirby, Biochim. Biophys. Acta 78, 732 (1963); Biochem. Prep. 11, 101 (1966). A solution of 350 mg monocyclohexylammonium enolpyruvate-P in 4 ml of water is mixed vigorously for 2 minutes with 2 ml of washed Dowex-50 (H+). The resin is removed by centrifugation and washed twice with 1.5 ml portions of water. The combined supernatant solutions are adjusted immediately to pH 6.8-7.0 with i N KOH, assayed by the method of Kornberg and Pricer, 5 and made up to 0.1 M. 5A. Kornberg and W. E. Pricer, Jr.,J. Biol. Chem. 193,481 (1951). sp. F. Knowles and D. B. Sprinson, this volume [43a].
[44]
PREPARATION OF ES-5-P
361
(to inhibit phosphatase activity), and 25 millimoles of Tris-chloride buffer, pH 7.2, is incubated at 37 ° for 30 minutes. The 0.4 to 0.6 saturated (NH4)2SO4fraction prepared from E. coli KI2 58-278 (25 ml, 20 mg of protein per ml) ~ is added, and incubation is continued for 1 hour. The reaction mixture is brought to pH 8 by the addition of 1 N NaOH, heated for 5 minutes in a boiling water bath, and clarified by filtration. Assay of a sample (0.5 ml) for enoipyruvate-P 5 indicates the disappearance of approximately 80 % of this substrate.~ Dowex l-X8 (chloride form, 200-400 mesh) is allowed to stand overnight in 3 N HCI, washed until the washings are free of chloride, and thoroughly freed of fine particles. A column of resin is prepared 50 cm high and 3 cm in diameter, covered with a 0.5 cm layer of acid-washed (3 N HCI) Berkshire sand, and equilibrated with 0.01 M Tris-chloride buffer, pH 9.0 (by passagq of 3 liters of buffer overnight). The filtered incubation mixture is loaded on the column at a rate of 1.5 ml/minute, and the column is washed with 500 ml of 0.01 M Tris-chloride buffer, pH 9.0. Separation is effected by gradient elution at a rate of 1.5 ml/minute with a 5-liter reservoir of 0.6 M LiCI in 0.01 M Tris-chloride buffer, pH 9, and a constant volume mixing chamber containing 3 liters of the buffer without LiCI. Fractions (250, 15 ml each) are collected on a fraction collector and monitored automatically in an ultraviolet analyzer by measuring absorption at 240 m/z; the first 1000 ml of eluate may be collected in one batch. In a typical pattern, shikimate-5-P is found in fractions 90-100; enolpyruvylshikimate, in fractions 103-110; enolpyruvate-P, in fractions 114-125; and ES-5-P, in fractions 162-188. 2 The last three compounds are identified by heating in 0.1 N HCI in a boiling water bath for 10 minutes, and determining the resulting pyruvate as the 2,4-dinitrophenylhydrazone. s The combined fractions containing ES-5-P (350 micromoles) are concentrated to 50 ml by rotary evaporation under reduced pressure (bath temperature, 35°), and treated with 40 ml of 0.1 M barium acetate (6 times the theoretical quantity) and 270 ml of cold absolute ethanol. After standing overnight at 0°, the precipitate is removed by centrifugation, washed four times with 10 ml portions of 67% ethanol, and dried in a vacuum over P205. The yield is 810 mg~ containing 320 micromoles of the barium salt of ES-5-P. The crude product is stirred with 10 ml of water, the suspension is cleared by centrifugation, and the supernatant solution is removed with a pipette. The process of extraction is repeated seven more times. The extracts (approximately 80 ml) are combined, treated with 160 ml rThe yield of ES-5-P is only 50 to 60 % since some enolpyruvate-P is lost in side reactions. ST. E. Friedemann and G. E. Haugen,J. Biol. Chem. 147, 41.5 ( ! 94:3).
362
AROMATIC AMINO ACIDS
[45]
of absolute ethanol, and stored overnight at 0 °. The precipitate is collected by centrifugation, washed four times with 10 ml portions of cold 67 % ethanol, and dried in a vacuum over P205. The yield is 196 mg of white solid, containing 290 micromoles of the barium salt of ES-5-P. At 100 ° under reduced pressure, this salt shows a loss in weight of 5.4%; the theoretical loss for Ba2(ES-5-P) • 2 H 2 0 is 5.7%. On the assumption of a molecular weight of 630 for the dihydrate, the purity of the barium salt is 94%. This degree of purity is also indicated by an analysis for constituent groups. 2 The barium salt of ES-5-P (1.3 rag, 2.06 micromoles) is converted to the potassium salt. Shikimate (2.0 micromoles) is produced only after treatment with alkaline phosphatase followed by hydrolysis with dilute acid, indicating the absence of enolpyruvylshikimate and shikimate-5-P. Hydrolysis with dilute acid released 1.9 micromoles of pyruvate, and treatment with alkaline phosphatase released 2.0 micromoles of orthophosphate, which was otherwise absent. Assay for enolpyruvate-P 5 gave a negative result.
[45] Preparation of Chorismic Acid By F. GIBSON COOH
O---C OH
COOH
Chorismic acid
Chorismic acid [a-(5-carboxy-l,2-dihydro-2-hydroxyphenoxy)acrylic acid] has not been synthesized chemically, but is readily accumulated by cell suspensions of a multiply-blocked strain ofAerobacter aerogenes (62-1) with metabolic blocks in the biosynthetic pathways leading to tryptophan, phenylalanine, and tyrosine.' Because the organism used is capable of forming anthranilate from chorismate, the cells are grown in a medium containing tryptophan to repress anthranilate synthetase. 2 The cells are then transferred to an accumulation mixture from which the free acid is isolated. 2,a IM. I. Gibson a n d F. Gibson, Biochem..], 90, 248 (1964); see also Biochem. Prepn. 12, 94
(1968). 2F. (;ibson, Biochem..]. 90, 256 (1964). % M. Edwards a n d I J. M. J a c k m a n , Australian.]. (;hem. 18, 1227 (1965).