- Email: [email protected]
[46] Chorismate synthase: A bifunctional enzyme in Neurospora crassa
362
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[46]
[46] C h o r i s m a t e S y n t h a s e : A B i f u n c t i o n a l E n z y m e in N e u r o s p o r a crassa By FRANK H. GAERTNER
3-Enolpyruvylshikimate5-phosphate
NADPH FMN
~chorismate+ phosphate
Chorismate synthase catalyzes the/3 elimination of phosphate from the prearomatic intermediate 3-enolpyruvylshikimate 5-phosphate. This seemingly simple reaction appears to be catalyzed by a complex enzymatic process. Catalytic amounts of flavin in the form of FMN and reduced pyridine nucleotide in the form of NADPH are required for chorismate synthase activity. The flavoprotein can act independently as a diaphorase, transferring electrons from NADPH by way of flavin to an electron-accepting dye such as 2,6-dichloroindophenol. ~ In prokaryotes such as Escherichia colt~ and Bacillus subtilis 3 the diaphorase activity appears to be catalyzed by separate proteins. Whereas, in the eukaryote Neurospora crassa, chorismate synthase and the diaphorase activity appear to be catalyzed by a single protein/Although essential for activity, it is not known what role the flavoprotein plays in the chorismate synthase reaction.
Assay Method Principle. The product of the reaction, chorismate, is catalyzed to anthranilate, a fluorescent intermediate of the tryptophan pathway. The reaction is carried out in two steps. In the first step, the substrate, 3enolpyruvylshikimate 5-phosphate, is catalyzed to chorismate. In the second, chorismate is stoichiometrically converted to anthranilate which is then selectively extracted into organic solvent and quantified fluorometrically. Reaction I Reagents
Potassium phosphate, 1.0 M, pH 7.0 3-Enolpyruvylshikimate 5-phosphate, 4 5 mM NADPH, 5 mM 1 G. R. Welch, K. W. Cole, and F. H. Gaertner, Arch. Biochem. Biophys. 165, 505 0974). 2 H. Morell, M. J. Clark, P. F. Knowles, and D. B. Sprinson, J. Biol. Chem. 242, 82 (1967). 3 N. Hasan and E. W. Nester, J. Biol. Chem. ?,53, 4987 (1978). 4
3-enolpyruvylshikimate5-phosphateis synthesizedenzymaticallyfrom shikimate,depro-
METHODS IN ENZYMOLOGY,VOL. 142
Copyright © 1987by Academic Press, Inc. All rightsof reproductionin any form reserved.
[46]
CHORISMATESYNTFIASE
363
FMN, 0. I mM Bovine serum albumin, 10 mg/ml Procedure. Reaction mixtures contain 10/zl of each of the reagents for Reaction I plus the enzyme solution to be assayed in a total volume of 100 /zl. Reactions are incubated at 25 °, stopped at selected time points by the addition of 20/zl 1.0 N HCI, neutralized after 10 min at room temperature with 20/zl 1.0 N NaOH, and added to a second reaction mixture designed to convert chorismate to the fluorescent product, anthranilate.
Reaction H Reagents Potassium phosphate, 1.0 M, pH 7.0 M g S O 4 , 0. I M
L-Glutamine, 0.4 M Anthranilate synthase, 0.2 units/ml 1 Procedure. The neutralized mixture from Reaction I is added to a second mixture containing 10/zl each of the reagents for Reaction II and 60/zl water. After 1 hr incubation the reaction is acidified by the addition of 20/zl of 2 N HCI and anthranilate is extracted with 0.4 ml ethyl acetate. Fluorescence of the organic phase is recorded in a spectrophotofluorometer at 340 nm excitation and 400 nm emission wavelengths. A more convenient but less accurate assay can be accomplished by following the appearance of anthranilate from 3-enolpyruvylshikimate 5-phosphate continuously in the spectrophotofluorometer. Definition of Unit. The formation of 1.0/xmol of anthranilate per min at 25° is defined as 1 unit of activity. The fluorescence of a standard solution of anthranilate extracted with ethyl acetate in the same manner as done for the assay samples is used at the time of each assay for the purpose of converting fluorescence to micromoles of product formed. Purification Procedure Wild type Neurospora crassa (strain 74A) is grown under forced aeration in Vogel's minimal medium 7 and harvested by trapping the mycelium in cheese cloth as previously described: teinated, and used without further purification. 5 It also can be chemically synthesized from shikimate 5-phosphate and used in purified form. 6 It should be noted that the enzymatically synthesized substrate contains divalent magnesium which may mask a requirement for this i o n ) 5 F. H. Gaertner and K. W. Cole, J. Biol. Chem. 248, 4602 (1973). 6 p. F. Knowles, J. G. Levin, and D. B. Sprinson, this series, Vol. 17A, p. 360. 7 H. J. Vogel, Microb. Genet. Bull. 13, 42 (1956). s F. H. Gaertner and J. A. DeMoss, this series, Vol. 17A, p. 387.
364
B I O S Y N T H E S I S OF T H E A R O M A T I C A M I N O ACIDS
[46]
Step 1. Crude extracts are prepared by suspending 150 g powdered lyophilized mycelium in 2100 ml of 0.05 M potassium phosphate buffer, pH 7.0, plus 10/~M FMN, 0.1 mM EDTA, and 0.2 mM DTT (0.05 M KEFD, pH 7.0). The suspension is stirred at 4° for 30 min, debris is removed by centrifugation at 10,000 g for 30 min, and 280 ml of 1.5% protamine sulfate solution is added dropwise, the suspension is stirred 15 min more, and the precipitate is removed by centrifugation at 10,000 g for 15 min. Step 2. Ammonium sulfate fractionation is accomplished by the gradual addition of 0.242 g of ammonium sulfate to each ml of crude extract. The solution is stirred for 30 min, the precipitate is removed by centrifugation at 10,000 g for 15 min, and 0.063 g ammonium sulfate is added to each ml of supernatant. The solution is mixed for 30 min and the precipitate is recovered by centrifugation at 10,000 g for 15 min. The precipitate is dissolved in 0.05 M KEFD buffer, pH 7.0, to yield a final volume of 60 ml. Step 3. The entire 60 ml volume from the previous step is passed through a 4 × 90 cm column of coarse-grade Sephadex G-25 equilibrated at 4 ° with 0.05 M KEFD, pH 7.0. The excluded fraction, which is brown in color, is collected. Step 4. The excluded fraction from step 3 is fractionated on a 2 x 80 cm column of DEAE-cellulose. The column is equilibrated with 0.01 M KEFD, pH 6.5, and eluted with a 1500 ml linear gradient of 0.01 to 0.15 M KEFD, pH 6.5. Fractions of 9.0 ml each are collected at 10 min intervals. Active fractions from about 110 through 160 are pooled and concentrated to 10 ml with ammonium sulfate at 70% saturation. Step 5. The 10 ml concentrated sample from step 6 is passed through a 2 × 50 cm column of coarse-grade Sephadex G-25 equilibrated and eluted at 4 ° with 0.01 M KEFD, pH 6.5. Step 6. The excluded fraction from step 5 is applied to a 2 x 80 cm column of phosphocellulose equilibrated with 0.01 M KEFD, pH 6.5. The column is eluted at 37 ml/hr with a 1500 ml linear gradient of 0.01 to 0.15 M KEFD, pH 6.5, followed by 300 to 500 ml wash of 0.15 M KEFD, pH 6.5, and a second 1500 ml linear gradient of 0.15 to 0.5 M KEFD, pH 6.5. The purified enzyme, approximately located in fractions 110 to 140, is pooled, concentrated by precipitation with ammonium sulfate, dissolved in 1.0 ml 0.1 M KEFD, pH 7.0, and stored frozen in liquid nitrogen. Recovery of chorismate synthase with this procedure is between 10 and 20%. Therefore, starting with approximately 30 units of enzyme activity in a crude extract from 150 g lyophilized mycelium, one can expect 3 to 6 units of purified enzyme with a specific activity of about 0.7 units per mg (assuming 1.0 mg/A280). PhosphoceUulose has specific affinity for
[46]
CHORISMATESYNTHASE
365
chorismate synthase 9 and serves well as a final purification step for the enzyme. Phosphocellulose has also been used to purify chorismate synthase from Bacillus subtilis. 3 As an alternative purification step, the enzyme from N . crassa has been purified by gel electrophoresis following DEAE-cellulose chromatography. 1 Properties Catalytic Properties. The catalytic properties of homogeneous preparations of chorismate synthase are markedly different from those found in preparations having purity of 50% or less. For example, with the purified enzyme F M N is required for activity, bovine serum albumin is needed to sustain the reaction, and the substrate 3-enolpyruvylshikimate 5-phosphate serves as an activator. Depending on the state of purity, these properties are either not seen or are much less pronounced. Specifically, with purified preparations the Km for FMN can be shown to be 1.0/zM, and FMN at l0/zM is optimal with higher concentrations of FMN inhibitory. Less pure preparations show no such requirement for FMN. Bovine serum albumin in the reaction mixture at 1 mg/ml appears to stabilize the purified enzyme since without added protein in the assay mixture, activity begins to decay within l0 min. Less pure preparations are not affected by the addition of this protein. At low concentrations of FMN, between 0.1 and 1.0/xM, chorismate synthase exhibits a 20 to 40 min lag. By incubating the pure enzyme for about 15 min at room temperature with 3enolpyruvylshikimate 5-phosphate in the absence of flavin and NADPH, catalysis does not take place, but in subsequent assay in the complete reaction mixture the lag is eliminated. Similar lags in the chorismate synthase reaction have been observed with the enzyme from Bacillus subtilis. 3
In addition to the/3 elimination of phosphate from 3-enolpyruvylshikimate 5-phosphate, chorismate synthase can also catalyze a flavin reductase or diaphorase-type reaction. An assay mixture for the diaphorase reaction contains potassium phosphate, 100 mM (pH 8), NADPH, 0.2 mM, FMN, 0.01 mM, 2,6-dichloroindophenol, 0.05 mM, and enzyme solution. Activity is measured by decrease in absorbance at 600 nm with a molar extinction coefficient for 2,6-dichloroindophenol of 21,000. A natural form of flavin reductase for chorismate synthase in E. coli has not been identified, but FAD and NAD requiring pig heart diaphorase can be used to replace this functional requirement for chorismate synthase. In B. subtilis a magnesium ion, NADPH, FAD, or FMN-dependent flavin reduc9 K. W. Cole and F. H. Gaertner, Biochern. Biophys. Res, Comrnun. 67, 170 (1975).
366
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[47]
tase has been identified as a separate enzyme that is required for the activity of chorismate synthase from this source? Physical Properties. Sucrose density gradient analyses of chorismate synthase indicate that the enzyme can exist in at least two active forms with sedimentation values of approximately 8 S and 10 S. 5 From SDS gel electrophoresis of the purified enzyme a single band with a molecular weight of 55,000 is obtained.l These results are consistent with the idea that the 8 S form of chorismate synthase is a dimer of the 55,000 molecular weight subunit and that the 10 S form is a tetramer. Since both the flavin reductase and the chorismate synthase activity appear to be catalyzed by a single protein, it is likely that in N. crassa chorismate synthase exists as a bifunctional enzyme. In contrast in E. coh~ and B. subtilis, 3 as has already been noted, flavin reductase and chorismate synthase are separable enzymatic activities.
[47] A n t h r a n i l a t e S y n t h a s e - A n t h r a n i l a t e Phosphoribosyltransferase Complex and Subunits of Salmonella t y p h i m u r i u m B y R O N A L D B A U E R L E , J O H N H E S S , and SARAH FRENCH
Introduction In Salmonella typhimurium and a number of other enteric bacteria, including Escherichia coli and Enterobacter aerogenes, the first two specific steps of tryptophan biosynthesis are catalyzed by a multifunctional allosteric enzyme, the anthranilate synthase-anthranilate 5-phosphorylribose-l-pyrophosphate phosphoribosyltransferase (AS-PRT) complex (Volume XVIIA [46,47,48a]). The A S - P R T complex is a tetramer made up of two molecules each of subunits component I and component II, the products of trpE and trpD, the first two genes of the trp oper0n.I The intact A S - P R T complex, 2 an AS partial complex derived by proteolytic digestion of the intact complex,3,4 and the uncomplexed subunits obtained from trpE and trpD mutant strains 5,6 have been purified and well characI R. Bauerle and P. Margolin, Cold Spring Harbor Symp. Quant. Biol. 31, 203 (1966). E. J. H e n d e r s o n and H. Zalkin, J. Biol. Chem. 246, 6891 (1971). 3 L. H. H w a n g and H. Zalkin, J. Biol. Chem. 246, 2338 (1971). 4 H. T a m i r and P. R. Srinivasan, J. Biol. Chem. 244, 6507 (1969). 5 H. Zalkin and D. Kling, Biochemistry 7, 3566 (1968). 6 M. G r i e s h a b e r and R. Bauerle, Biochemistry 13, 373 (1974).
METHODS IN ENZYMOLOGY, VOL. 142
Copyright © 1987by AcademicPress, Inc.
All rights of reproduction in any form reserved.
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[46]
[46] C h o r i s m a t e S y n t h a s e : A B i f u n c t i o n a l E n z y m e in N e u r o s p o r a crassa By FRANK H. GAERTNER
3-Enolpyruvylshikimate5-phosphate
NADPH FMN
~chorismate+ phosphate
Chorismate synthase catalyzes the/3 elimination of phosphate from the prearomatic intermediate 3-enolpyruvylshikimate 5-phosphate. This seemingly simple reaction appears to be catalyzed by a complex enzymatic process. Catalytic amounts of flavin in the form of FMN and reduced pyridine nucleotide in the form of NADPH are required for chorismate synthase activity. The flavoprotein can act independently as a diaphorase, transferring electrons from NADPH by way of flavin to an electron-accepting dye such as 2,6-dichloroindophenol. ~ In prokaryotes such as Escherichia colt~ and Bacillus subtilis 3 the diaphorase activity appears to be catalyzed by separate proteins. Whereas, in the eukaryote Neurospora crassa, chorismate synthase and the diaphorase activity appear to be catalyzed by a single protein/Although essential for activity, it is not known what role the flavoprotein plays in the chorismate synthase reaction.
Assay Method Principle. The product of the reaction, chorismate, is catalyzed to anthranilate, a fluorescent intermediate of the tryptophan pathway. The reaction is carried out in two steps. In the first step, the substrate, 3enolpyruvylshikimate 5-phosphate, is catalyzed to chorismate. In the second, chorismate is stoichiometrically converted to anthranilate which is then selectively extracted into organic solvent and quantified fluorometrically. Reaction I Reagents
Potassium phosphate, 1.0 M, pH 7.0 3-Enolpyruvylshikimate 5-phosphate, 4 5 mM NADPH, 5 mM 1 G. R. Welch, K. W. Cole, and F. H. Gaertner, Arch. Biochem. Biophys. 165, 505 0974). 2 H. Morell, M. J. Clark, P. F. Knowles, and D. B. Sprinson, J. Biol. Chem. 242, 82 (1967). 3 N. Hasan and E. W. Nester, J. Biol. Chem. ?,53, 4987 (1978). 4
3-enolpyruvylshikimate5-phosphateis synthesizedenzymaticallyfrom shikimate,depro-
METHODS IN ENZYMOLOGY,VOL. 142
Copyright © 1987by Academic Press, Inc. All rightsof reproductionin any form reserved.
[46]
CHORISMATESYNTFIASE
363
FMN, 0. I mM Bovine serum albumin, 10 mg/ml Procedure. Reaction mixtures contain 10/zl of each of the reagents for Reaction I plus the enzyme solution to be assayed in a total volume of 100 /zl. Reactions are incubated at 25 °, stopped at selected time points by the addition of 20/zl 1.0 N HCI, neutralized after 10 min at room temperature with 20/zl 1.0 N NaOH, and added to a second reaction mixture designed to convert chorismate to the fluorescent product, anthranilate.
Reaction H Reagents Potassium phosphate, 1.0 M, pH 7.0 M g S O 4 , 0. I M
L-Glutamine, 0.4 M Anthranilate synthase, 0.2 units/ml 1 Procedure. The neutralized mixture from Reaction I is added to a second mixture containing 10/zl each of the reagents for Reaction II and 60/zl water. After 1 hr incubation the reaction is acidified by the addition of 20/zl of 2 N HCI and anthranilate is extracted with 0.4 ml ethyl acetate. Fluorescence of the organic phase is recorded in a spectrophotofluorometer at 340 nm excitation and 400 nm emission wavelengths. A more convenient but less accurate assay can be accomplished by following the appearance of anthranilate from 3-enolpyruvylshikimate 5-phosphate continuously in the spectrophotofluorometer. Definition of Unit. The formation of 1.0/xmol of anthranilate per min at 25° is defined as 1 unit of activity. The fluorescence of a standard solution of anthranilate extracted with ethyl acetate in the same manner as done for the assay samples is used at the time of each assay for the purpose of converting fluorescence to micromoles of product formed. Purification Procedure Wild type Neurospora crassa (strain 74A) is grown under forced aeration in Vogel's minimal medium 7 and harvested by trapping the mycelium in cheese cloth as previously described: teinated, and used without further purification. 5 It also can be chemically synthesized from shikimate 5-phosphate and used in purified form. 6 It should be noted that the enzymatically synthesized substrate contains divalent magnesium which may mask a requirement for this i o n ) 5 F. H. Gaertner and K. W. Cole, J. Biol. Chem. 248, 4602 (1973). 6 p. F. Knowles, J. G. Levin, and D. B. Sprinson, this series, Vol. 17A, p. 360. 7 H. J. Vogel, Microb. Genet. Bull. 13, 42 (1956). s F. H. Gaertner and J. A. DeMoss, this series, Vol. 17A, p. 387.
364
B I O S Y N T H E S I S OF T H E A R O M A T I C A M I N O ACIDS
[46]
Step 1. Crude extracts are prepared by suspending 150 g powdered lyophilized mycelium in 2100 ml of 0.05 M potassium phosphate buffer, pH 7.0, plus 10/~M FMN, 0.1 mM EDTA, and 0.2 mM DTT (0.05 M KEFD, pH 7.0). The suspension is stirred at 4° for 30 min, debris is removed by centrifugation at 10,000 g for 30 min, and 280 ml of 1.5% protamine sulfate solution is added dropwise, the suspension is stirred 15 min more, and the precipitate is removed by centrifugation at 10,000 g for 15 min. Step 2. Ammonium sulfate fractionation is accomplished by the gradual addition of 0.242 g of ammonium sulfate to each ml of crude extract. The solution is stirred for 30 min, the precipitate is removed by centrifugation at 10,000 g for 15 min, and 0.063 g ammonium sulfate is added to each ml of supernatant. The solution is mixed for 30 min and the precipitate is recovered by centrifugation at 10,000 g for 15 min. The precipitate is dissolved in 0.05 M KEFD buffer, pH 7.0, to yield a final volume of 60 ml. Step 3. The entire 60 ml volume from the previous step is passed through a 4 × 90 cm column of coarse-grade Sephadex G-25 equilibrated at 4 ° with 0.05 M KEFD, pH 7.0. The excluded fraction, which is brown in color, is collected. Step 4. The excluded fraction from step 3 is fractionated on a 2 x 80 cm column of DEAE-cellulose. The column is equilibrated with 0.01 M KEFD, pH 6.5, and eluted with a 1500 ml linear gradient of 0.01 to 0.15 M KEFD, pH 6.5. Fractions of 9.0 ml each are collected at 10 min intervals. Active fractions from about 110 through 160 are pooled and concentrated to 10 ml with ammonium sulfate at 70% saturation. Step 5. The 10 ml concentrated sample from step 6 is passed through a 2 × 50 cm column of coarse-grade Sephadex G-25 equilibrated and eluted at 4 ° with 0.01 M KEFD, pH 6.5. Step 6. The excluded fraction from step 5 is applied to a 2 x 80 cm column of phosphocellulose equilibrated with 0.01 M KEFD, pH 6.5. The column is eluted at 37 ml/hr with a 1500 ml linear gradient of 0.01 to 0.15 M KEFD, pH 6.5, followed by 300 to 500 ml wash of 0.15 M KEFD, pH 6.5, and a second 1500 ml linear gradient of 0.15 to 0.5 M KEFD, pH 6.5. The purified enzyme, approximately located in fractions 110 to 140, is pooled, concentrated by precipitation with ammonium sulfate, dissolved in 1.0 ml 0.1 M KEFD, pH 7.0, and stored frozen in liquid nitrogen. Recovery of chorismate synthase with this procedure is between 10 and 20%. Therefore, starting with approximately 30 units of enzyme activity in a crude extract from 150 g lyophilized mycelium, one can expect 3 to 6 units of purified enzyme with a specific activity of about 0.7 units per mg (assuming 1.0 mg/A280). PhosphoceUulose has specific affinity for
[46]
CHORISMATESYNTHASE
365
chorismate synthase 9 and serves well as a final purification step for the enzyme. Phosphocellulose has also been used to purify chorismate synthase from Bacillus subtilis. 3 As an alternative purification step, the enzyme from N . crassa has been purified by gel electrophoresis following DEAE-cellulose chromatography. 1 Properties Catalytic Properties. The catalytic properties of homogeneous preparations of chorismate synthase are markedly different from those found in preparations having purity of 50% or less. For example, with the purified enzyme F M N is required for activity, bovine serum albumin is needed to sustain the reaction, and the substrate 3-enolpyruvylshikimate 5-phosphate serves as an activator. Depending on the state of purity, these properties are either not seen or are much less pronounced. Specifically, with purified preparations the Km for FMN can be shown to be 1.0/zM, and FMN at l0/zM is optimal with higher concentrations of FMN inhibitory. Less pure preparations show no such requirement for FMN. Bovine serum albumin in the reaction mixture at 1 mg/ml appears to stabilize the purified enzyme since without added protein in the assay mixture, activity begins to decay within l0 min. Less pure preparations are not affected by the addition of this protein. At low concentrations of FMN, between 0.1 and 1.0/xM, chorismate synthase exhibits a 20 to 40 min lag. By incubating the pure enzyme for about 15 min at room temperature with 3enolpyruvylshikimate 5-phosphate in the absence of flavin and NADPH, catalysis does not take place, but in subsequent assay in the complete reaction mixture the lag is eliminated. Similar lags in the chorismate synthase reaction have been observed with the enzyme from Bacillus subtilis. 3
In addition to the/3 elimination of phosphate from 3-enolpyruvylshikimate 5-phosphate, chorismate synthase can also catalyze a flavin reductase or diaphorase-type reaction. An assay mixture for the diaphorase reaction contains potassium phosphate, 100 mM (pH 8), NADPH, 0.2 mM, FMN, 0.01 mM, 2,6-dichloroindophenol, 0.05 mM, and enzyme solution. Activity is measured by decrease in absorbance at 600 nm with a molar extinction coefficient for 2,6-dichloroindophenol of 21,000. A natural form of flavin reductase for chorismate synthase in E. coli has not been identified, but FAD and NAD requiring pig heart diaphorase can be used to replace this functional requirement for chorismate synthase. In B. subtilis a magnesium ion, NADPH, FAD, or FMN-dependent flavin reduc9 K. W. Cole and F. H. Gaertner, Biochern. Biophys. Res, Comrnun. 67, 170 (1975).
366
BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS
[47]
tase has been identified as a separate enzyme that is required for the activity of chorismate synthase from this source? Physical Properties. Sucrose density gradient analyses of chorismate synthase indicate that the enzyme can exist in at least two active forms with sedimentation values of approximately 8 S and 10 S. 5 From SDS gel electrophoresis of the purified enzyme a single band with a molecular weight of 55,000 is obtained.l These results are consistent with the idea that the 8 S form of chorismate synthase is a dimer of the 55,000 molecular weight subunit and that the 10 S form is a tetramer. Since both the flavin reductase and the chorismate synthase activity appear to be catalyzed by a single protein, it is likely that in N. crassa chorismate synthase exists as a bifunctional enzyme. In contrast in E. coh~ and B. subtilis, 3 as has already been noted, flavin reductase and chorismate synthase are separable enzymatic activities.
[47] A n t h r a n i l a t e S y n t h a s e - A n t h r a n i l a t e Phosphoribosyltransferase Complex and Subunits of Salmonella t y p h i m u r i u m B y R O N A L D B A U E R L E , J O H N H E S S , and SARAH FRENCH
Introduction In Salmonella typhimurium and a number of other enteric bacteria, including Escherichia coli and Enterobacter aerogenes, the first two specific steps of tryptophan biosynthesis are catalyzed by a multifunctional allosteric enzyme, the anthranilate synthase-anthranilate 5-phosphorylribose-l-pyrophosphate phosphoribosyltransferase (AS-PRT) complex (Volume XVIIA [46,47,48a]). The A S - P R T complex is a tetramer made up of two molecules each of subunits component I and component II, the products of trpE and trpD, the first two genes of the trp oper0n.I The intact A S - P R T complex, 2 an AS partial complex derived by proteolytic digestion of the intact complex,3,4 and the uncomplexed subunits obtained from trpE and trpD mutant strains 5,6 have been purified and well characI R. Bauerle and P. Margolin, Cold Spring Harbor Symp. Quant. Biol. 31, 203 (1966). E. J. H e n d e r s o n and H. Zalkin, J. Biol. Chem. 246, 6891 (1971). 3 L. H. H w a n g and H. Zalkin, J. Biol. Chem. 246, 2338 (1971). 4 H. T a m i r and P. R. Srinivasan, J. Biol. Chem. 244, 6507 (1969). 5 H. Zalkin and D. Kling, Biochemistry 7, 3566 (1968). 6 M. G r i e s h a b e r and R. Bauerle, Biochemistry 13, 373 (1974).
METHODS IN ENZYMOLOGY, VOL. 142
Copyright © 1987by AcademicPress, Inc.
All rights of reproduction in any form reserved.