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[71] Tyrosine and phenylalanine biosynthesis: The T and P proteins (Aerobacter aerogenes); chorismate mutase (Pisum sativum)
564
AROMATIC AMINO ACIDS
[71 ]
[71] Tyrosine and Phenylalanine Biosynthesis: The T and P Proteins (Aerobacter aerogenes); Chorismate Mutase (Pisum sativum)
By R.
G. H. COTTON and F. GIBSON Introduction
The terminal reactions in the biosynthesis of tyrosine and phenylalanine involve the conversion of chorismate through prephenate to 4-hydroxyphenylpyruvate or phenylpyruvate, which are then transaminated to give the amino acids. 1 Although the intermediates are the Phenylpyruvate
~ Phenylalanine
/2
(prephenate)
/1
Chorismate
\3
(prephenate)
\,
4- Hydroxyphenylpyruvate
-'-- Tyrosine
(a)
Phenylpyruvate ~
Phenylalanine
/ Chorismate - -
Prephenate
\
4- Hydroxyphenylpyruvate
~
Tyrosine
(b) FIG. 1. Pathways for the conversion of chorismate to phenylalanine and tyrosine (see text footnote 1). (a) T h e pathways in Aerobacter aerogenes and Escherichia coll. T h e P protein carries out reaction 1 (chorismate mutase P activity) and reaction 2 (prephenate dehydratase activity). T h e T protein carries out reaction 3 (chorismate mutase T activity) and reaction 4 (prephenate dehydrogenase activity). (b) T h e pathways in a n u m b e r of other organisms, e.g., Neurospora and Pisum sativum (the pea). IF. Gibson and .l. Pittard, Bacteriol. Rev. 32,465 (1968).
[71]
TYROSINE AND PHENYLALANINE BIOSYNTHESIS
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same in different types of cells, details of the pathways vary. In Aerobacter aerogenes and in Escherichia coli the conversion of chorismate to 4hydroxyphenylpyruvate is carried out by a single protein or protein complex. 2-4 There is evidence that conversion of chorismate to phenylpyruvate is also carried out by a single protein. 2 These pathways are shown in Fig. la. In some other organisms ~ (see also Table II) only one chorismate mutase activity is present and the pathways branch at prephenate (Fig. 1b).
T Protein Assay Methods Principle. Two reactions are carried out by the T protein: COOH HOOC
/CH2--C--COOH
chorismate mutase
OH
COOH
0
OH
Chorismic acid
Prephenic acid + DPN~prephenat e
~///
dehydrogenase
CH2--C--COOH o + DPNH + H÷ + CO2 OH
O f the two activities carried out by the T protein, chorismate mutase is more readily assayed in crude cell extracts. 5 Chorismate mutase P (see later) is also present in crude cell extracts. Chorismate mutase activity is assayed by estimating the prephenate formed. Prephenate dehydrogenase activity is measured by a fluorometric determination of the DPNH formed during the conversion of prephenate into 4-hydroxyphenylpyruvate. 2R. G. H. Cotton and F. Gibson, Biochim. Biophys..4eta 100, 76 (1965). nR. G. H. Cotton and F. Gibson, Biochim. Biophys. Acta 147,222 (1967). +R. G. H. Cotton and F. Gibson, Biochim. Biophys. Acta 160, 188 (1968). °Prephenate dehydrogenase may be assayed in crude extracts by a modified phenol assay as described by I. Schwinck and E. Adams, Biochim. Biophys. Acta 36, 102 (1959).
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AROMATICAMINOACIDS
[71]
Reagents Chorismic acid, 0.01 M Potassium prephenate, 0.01 M Dithiothreitol, 0.01 M EDTA, 0.001 M Tris-HCl buffer, pH 8.2, 0.5 M DPN +, 0.05 M HC1, M NaOH, M Enzyme
Procedure for Chorismate Mutase Assay Step 1. The incubation mixture contains in a final volume of 1.0 ml: 0.1 ml of chorismic acid, 0.1 ml of dithiothreitol, 0.1 ml of EDTA, 0.1 ml of Tris-HC1 buffer, and about 3 units of enzyme. After incubation for 20 minutes at 37 °, the reaction is stopped by plunging the tubes into an ice bath. Step 2. Prephenate is measured in the incubated samples and in a zero time control by estimating phenylpyruvate formed from prephenate by acid treatment of the samples. From each tube two 0.4-ml samples (A and B) are taken. To A is added 0.4 ml of HCI arid tke mixture incubated 10 minutes at 37 ° after which 3.2 ml of NaOH is added and the absorbance at 320 m/x is measured. To B is added 3.2 ml of N a O H followed by 0.4 ml of HCI, and the absorbance at 320 m/x is measured. The difference in absorbance at 320 m/z between A and B indicates the amount of phenylpyruvate formed from prephenate. Phenylpyruvate has a molar absorption coefficient of 17,500 in alkaline solution. The absorbance is measured as soon as possible after the addition of alkali, as the chromophore is unstable, n In each experiment a control without enzyme is included to estimate the small amount of prephenate formed nonenzymatically from chorismate during the incubation period. Chorismate mutase may also be assayed by following the disappearance of chorismate spectrophotometrically? Definition of Unit and Specific Activity. One unit of enzyme activity is defined as the amount catalyzing the formation of 0.1 micromole of prephenate in 20 minutes at 37 °. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. T eC. H. Doy and F. Gibson, Biochim. Biophys. Acta 50, 495 (1961). 70. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see also Vol. III [73].
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TYROSINE AND PHENYLALANINE BIOSYNTHESIS
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Procedure for Prephenate Dehydrogenase Assay The incubation mixture contains in a final volume of 1.5 ml in a quartz cuvette: 0.075 ml of potassium prephenate., 0.075 ml of Tris-HC1 buffer, 0.03 ml of DPN +, 0.15 ml of dithiothreitol, 0.15 ml of EDTA, and about 3 units of enzyme. The reaction mixture without DPN + is incubated for 3 minutes in a 37 ° water bath and then transferred to a temperature-controlled (37 °) compartment of an Aminco-Bowman, or similar spectrophotofluorometer. The reaction is started by the addition of DPN +, and the initial rate of increase of fluorescence (340 m/~ activation, 440 m/~ fluorescence; uncorrected) is measured on an X-Y recorder for about 4 minutes. The concentration of DPNH formed is measured using a standard curve prepared by measuring the fluorescence of known concentrations of DPNH. Purified preparations of prephenate dehydrogenase may also be measured spectro photometrically. 3 Definition of Unit and Specific Activity. One unit of enzyme activity is taken as that amount catalyzing the formation of 0.1 micromole of DPNH in 20 minutes at 37 °. Specific activity is expressed in units per milligram of protein. Protein is determined by the procedure of Lowry
et al.7 Purification Procedure A multiple aromatic auxotroph of .4. aerogenes (strain poly 3 was used in the purification described) TM is grown in a medium in which growth is limited by tyrosine, thus derepressing the T protein about 3-fold with respect to the wild-type strain grown in minimal medium. Several buffers are used: Buffer A: 0.01 M potassium phosphate (pH 7.0) containing 10 -3 M mercaptoethanol and 10 -4 M EDTA. Buffer B: as for buffer A, except pH 6.0 Buffer C: 0.025 M potassium phosphate (pH 8.0) containing 10-3 M mercaptoethanol and 10-4 M EDTA Buffer D: 0.1 M citric acid-potassium phosphate (pH 3.8) Buffer E: 0.2 M potassium phosphate (pH 8.0) containing 10-a M mercaptoethanol, 10-4 M EDTA and 0.3 M sodium chloride Buffer F: as for buffer A, but including 0.05 M sodium chloride
Step 1. Growth of Cells and Preparation of Cell-Free Extract. Twenty nutrient agar slopes of.4. aerogenes poly 3 are grown overnight at 37 ° and ~aAerobacter aerogenes poly 3 may be obtained from F. Gibson, Biochemistry Department,
I.A.S. Australian National University, Canberra, A.C.T., Australia.
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AROMATIC AMINOACIDS
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the growth is washed off with sterile glucose solution as inoculum for 40 liters of culture medium (glucose (0.16%)-citrate-mineral salts medium s containing 10-4 M L-tryptophan, 2 × 10-4 M L-phenylalanine, 10-n M p-aminobenzoic acid, and 9 × 10-5 M L-tyrosine). The culture is stirred and aerated at 37 ° until 1 hour after growth ceases. T h e absorbance of the fully grown culture is 1.5 at 600 m/~ in a 1-cm cell. The culture is stored at 0-4 ° overnight, and the cells are harvested by centrifugation. The cells are then washed by suspending them in about five times their packed volume of 0.9% NaCI, then centrifuging at 4 ° for 15 minutes at 10,000 g. T h e cells are then suspended in 4 ml of buffer A for each gram wet weight and smashed in a Ribi cell disintegrator at 20,000 psi (or French press). Whole cells and cell debris are removed by centrifugation at 17,000 g for 20 minutes. The supernatant (about 200 ml) is decanted and stored at--20 °. All the subsequent steps are carried out at 0-4 °. Step 2. Removal of Nucleic Acid. To each 200 ml of crude extract is added 33.3 ml of neutralized 0.4% protamine sulfate, and the mixture is stirred for 20 minutes. After centrifugation at 17,000 g for 20 minutes, the supernatant is decanted. Step 3. Treatment at pH 5.5. An equal volume of cold buffer D is added rapidly to the supernatant from step 2, and the mixture is stirred for 70 minutes. The final pH is about 5.5. After centrifugation at t2,000 g for 20 minutes, the supernatant is discarded and the precipitate is resuspended in an equal volume of buffer E and stirred for 1 hour. The supernatant obtained after a further centrifugation at 12,000 g for 20 minutes is used for the next step. Step 4. Ammonium Sulfate Treatment. Solid ammonium sulfate is added over a period of 15 minutes to give 40% saturation, and the solution stirred for a further 30 minutes. The precipitate which is sedimented at 12,000 g for 20 minutes is suspended in the minimum volume of buffer F and then dialyzed twice against 2 liters of buffer F for 1 hour. Step 5. Sephadex Chromatography. The dialyzed solution (up to 40 ml) is then applied to a 4.2 × 115 cm column of Sephadex G-100 and eluted with buffer F. The active fractions are pooled. Step 6. DEAE-Cellulose Chromatography. The pooled active fractions are applied to a 2.6 x 20 cm DEAE-cellulose column equilibrated with buffer A. The sample is washed in with 20 ml of buffer A containing 0.1 M NaC1. The column is then treated by linear gradient etution starting with 500 ml of buffer A containing 0.1 M NaC1 in the mixing bottle and 500 ml of the same buffer containing 0.2 M NaCI in the inlet SH. J. Vogel, and D. M. Bonner,J. Biol. Chem. 218, 97 (1956); see also this volume [ 1 ].
[71]
TYROSINE AND PHENYLALANINE BIOSYNTHESIS
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bottle. Elution is completed by additional buffer A containing 0.2 M NaCI. Fractions are collected every 16 minutes with a flow rate of 75 ml per hour. Chorismate mutase P appears at the front and is discarded, whereas the T protein appears in fractions 36-56. Step 7. Concentration by DEAE-Cellulose Chromatography. The active fractions (other than chorismate mutase P) are pooled and diluted with an equal volume of buffer A; the enzyme is concentrated on two 2 x 13 cm DEAE-cellulose columns equilibrated with buffer A. Half of the enzyme solution is applied to each of the columns at about 250 ml per hour, and then the adsorbed protein is quickly eluted with buffer E. The active fractions at the protein front are pooled and dialyzed three times for 1 hour against 2 liters of buffer B. Step 8. Chromatography on Brushite. The dialyzed sample is applied to a 2.6 x 20 cm column of freshly decanted Brushite 9 equilibrated with buffer B. After washing in the sample with buffer B, a linear gradient is applied starting with 400 ml of buffer B in the mixing flask and 400 ml of buffer C in the inlet flask. The protein is eluted with buffer at 75 mi per hour; fractions are collected at 16-minute intervals and the elution is completed with further buffer C. The T protein appears in fractions 46-64. Step 9. Concentration by DEAE-Cellulose Chromatography. The active fractions are pooled and applied to a 1.5 x 10 cm DEAE-cellulose column at 200 ml per hour. Protein is then quickly eluted with buffer E most of the protein being collected in less than 5 ml. A summary of the purification is given in Table I. The loss of activity following elution from Brushite and its subsequent recovery following concentration on DEAE-cellulose are probably due to the reversible formation of inactive subunits? ,4
Properties Stability. Concentrated solutions of the enzyme appear to be stable on storage at - 2 0 °. About 5% loss of activity is observed on freezing and thawing. In dilute solutions at 4 °, activity is lost but may be largely recovered by concentration. Dialysis and freezing causes inactivation which is reversed by thiols. Purity. Polyacrylamide gel disc electrophoresis reveals one major band of protein, with several slower moving, and one faster moving, faint bands. The heavy band can be shown to correspond to the prephenate dehydrogenase activity using a dehydrogenase stain technique? ° 9A. Tiselius, S. Hjert6n, and O. Levin, Arch. Biochem. Biophys. 65, 132 (1956); see also Vol.
v[2]. 1°C. L. Markert and F. M~ller, Proc. Natl. Acad. Sci. U.S. 45,753 (1959).
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AROMATIC AMINO ACIDS
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TABLE I SUMMARYOF PURIFICATION PROCEDUREOF Y PROTEINa
Protein
Step
Fraction
1 2 3 4
Cell-free extract Protamine sulfate supernatant pH precipitate 0-40% Ammonium sulfate
200 200 196 30
578 392 201 1035
5 6 7
Sephadex chromatography DEAE-cellulosechromatography DEAE-cellulosechromatography (concentration) Brushite DEAE-cellulosechromatography
150 415 45
164 33 256 3.7 1037
8 9
Specific activity (units/mg
Volume Units/ml (mg/ml) protein)
320 2.5
22.6 12.4 3.9 14.4
Yield
(%)
26 32 52 72
100 68 34 27
0.62 0.07 0.41
265 470 625
21 12 10
0.012 0.38
310 2720
1 2.3
aThe purification is followed by measuring chorismate mutase activity; therefore values
for specific activity given for steps 1-4 are approximate because of the presence of some P protein (about 30% of the chorismate mutase activity in the crude cell extract), which is finallycompletely separated during step 6. Kinetic Properties of the T Protein. 3 Chorismate mutase T has a p H o p t i m u m at about 8.5 in Tris-HC1 and p r e p h e n a t e d e h y d r o g e n a s e has highest activity at p H values above 8.6. Reciprocal plots o f rate-concentration p a r a m e t e r s for chorismate mutase are usually n o n l i n e a r at low concentrations o f chorismate. At high concentrations the plots are linear, giving an a p p a r e n t Km for chorismate o f 5.6 × 10 -4 M. For p r e p h e n a t e d e h y d r o g e n a s e the app a r e n t Km for DPN is 1.5 × 10 -4 M and for p r e p h e n a t e 1.1 × 10 -4 M. T P N has about half the activity o f DPN as a cofactor. Inhibitors and Activators. 3 T h e e n d - p r o d u c t inhibitor, L-tyrosine at 10 -4 M, inhibits p r e p h e n a t e d e h y d r o g e n a s e 50%, but does not affect chorismate mutase T. P r e p h e n a t e d e h y d r o g e n a s e was inhibited 50% by 4 - h y d r o x y c i n n a m a t e , 4 - h y d r o x y p h e n y l p y r u v a t e , a n d 4 - h y d r o x y phenyllactate, each at 10 -3 M, a n d by D-tyrosine and 4 - h y d r o x y b e n z o a t e , each at 10 -2 M. T y r a m i n e is not an inhibitor. S o d i u m dodecyl sulfate or cupric sulfate inhibited 50% at 10 -5 M a n d 10 -4 M, respectively. Only the last two c o m p o u n d s affect chorismate mutase T, reversibly inactivating the enzyme. T h e effects o f inhibitors o n the kinetics have been discussed? Antibody against the T protein inhibits chorismate mutase activity u p to 95%. Molecular Weight and Subunit Structure. 4 T h e T protein has a molecular weight o f about 87,000 but is dissociable into subunits without choris-
[71 ]
TYROSINE AND PHENYLALAN1NEBIOSYNTHESIS
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mate mutase or prephenate dehydrogenase activity by treatment with either acid or sodium dodecyl sulfate. The subunits, which have a molecular weight of about 45,000, can be recombined to give active protein carrying out both of the reactions.
P Protein The P protein has not been extensively purified; a partial purification, on a small scale, together with some properties of the protein is described.
Assay Methods
Principle. The P protein has two enzymatic activities, namely, chorismate mutase (1) and prephenate dehydratase (2). Chorismate Prephenate
, prephenate
(1)
~ phenylpyruvate+ H20 + CO2
(2)
Chorismate mutase is assayed as described for the T protein. Prephenate dehydratase is assayed by estimating the phenylpyruvate formed from prephenate.
Reagents Potassium prephenate, 0.01 M Tris-HCl buffer, 0.25 M, pH 8.2 NaOH, M Enzyme
Procedure. The incubation mixture contains, in a final volume of 1 ml: 0.05 ml of potassium prephenate, 0.1 ml of Tris-HCl buffer, and about 3 units of enzyme. After incubation for 30 minutes at 37 °, 3.0 ml of N a O H is added and the absorbancy at 320 m/x is measured. Phenylpyruvate is estimated using a molar absorption coefficient of 17,500. An unincubated control is also assayed for phenylpyruvate. Definition of Unit and Specific Activity. One unit of enzyme activity is defined as the amount catalyzing the formation of 0.1 micromole of phenylpyruvate in 20 minutes at 37 °. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al/ Purification Procedure The partial purification of the P protein from a wild-type strain of
A. aerogenes (NCW) is described. The same method has also been used
572
AROMATIC AMINO ACIDS
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with the P protein from E. coil W and E. coil K12, as wall as another strain of A. aerogenes. Step 1. Preparation of Cell-Free Extract. Cells are grown in a citratemineral salts medium 8 supplemented with 0.16% glucose. One-liter quantities of medium are inoculated with 1 ml from a 5-hour nutrient broth culture and incubated with shaking at 37 °. Cells are harvested in the late logarithmic phase and collected by centrifugation at 12,000 g for 20 minutes. The cells are washed in 0.9% NaC1 (one-twelfth the original medium volume) and finally suspended in 4 ml of 0.1 M potassium phosphate buffer (pH 7.0) for each gram wet weight of cells. After disintegration in a French pressure cell at 20,000 psi, the unbroken cells and cell debris are removed by centrifugation at 17,000 g for 15 minutes. About 4 ml of cell-free extract is obtained from each liter of culture. The crude cell-free extract is stored at --20 °. Step 2. Chromatography on DEAE-Cellulose. The cell-free extract is dialyzed twice for 1 hour against 2 liters of 0.01 M potassium phosphate buffer (pH 7.0) (the column buffer), and 4 ml is applied to a 2 × 20 cm column of DEAE-cellulose previously equilibrated against the above buffer. After washing in the protein with 2 lots of 5 ml of the column buffer, the column is then treated by linear gradient elution with 400 ml of the column buffer in the mixing flask and 400 ml of the same buffer containing 0.5 M NaCI in the inlet bottle. The eluent is passed through the column at 150 ml per hour, and 7.5-ml fractions are collected. The P protein (chorismate mutase P and prephenate dehydratase activities) is eluted about fraction 33 and the T protein at about fraction 58. A'. second unstable prephenate dehydratase (A) is present in A. aerogenes NCW and is eluted at the protein front. Properties ~
Stability. The P protein from A. aerogenes is unstable at 4 ° and is protected against heat by phenylalanine. The protein from E. coli is more stable. Inhibitors. Prephenate dehydratase is completely inhibited by 10 -3 M L-phenylalanine and inhibited 4 4 % by 5..× 10 -3 M L-tyrosine. Chorismate mutase P is inhibited 6 5 % by 5 × 10 ~3 M I:phenyialanine and 16% by 5 × 10-3 M l:tyrosine. Prephenate dehydratase A is relatively unaffected by the amino acids. Chorismate mutase P is inhibited by high concent~rat4ons o f thiol reagents and also by ferrous ions and detergent. Chorismate Mutase from Peas (Pisum sativum) There appears to be only a single chorismate mutase 4n the pea
[71]
TYROSINE AND PHENYLALANINE BIOSYNTHESIS
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where the steps correspond to those shown in Fig. lb. The enzyme has been examined after chromatography on DEAE-cellulose n as described below. Assays are carried out as previously described for P protein. Step 1. Preparation of CeU-Free Extract. Seedlings are grown for 10 days in moist sterile absorbent cotton wool. A 10-g quantity of cotyledons is homogenized in a Waring blendor for 1 minute with 10 ml. of 0.1 M Tris-HCl buffer (pH 7.8) containing 0.025 M sodium thioglycolate. The extract is then filtered through gauze and centrifuged at 17,000 g for 20 minutes. After dialysis twice for 1 hour against 0.01 M potassium phosphate buffer (pH 7.0), the extract is centrifuged again; the supernatant is used for the next step. Step 2. Chromatography on DEAE-Cellulose. The enzyme solution is chromatographed on a 2 × 30 cm DEAE-cellulose column previously equilibrated with 0.01 M phosphate buffer (pH 7.0) (column buffer). The column is then treated with a linear gradient starting with 500 ml of the column buffer in the mixing flask and 500 ml of the same buffer containing 0.5 M NaCI in the inlet flask. The flow rate is 150 ml per hour, 10-ml fractions being collected every 4 minutes. Activity appears in fraction 39. Properties t 1
Inhibitors and Activators. DL-Tryptophan (1.2 X 10-4 M) stimulated the activity in crude cell extracts about 20% and in column fractions up to 3-fold. This concentration of tryptophan was included in all assays. L-Phenylalanine and L-tyrosine both inhibit the enzyme, their effects being antagonized by DL-tryptophan. Related Enzymes. Prephenate dehydrogenase or prephenate dehydratase were not detectable in extracts of pea cotyledons prepared as described. TM Distribution o f Enzymatic Activities Related to Chorismate and Prephenate Metabolism
The enzymes described are widely distributed, probably being present in all organisms which synthesize their own phenylalanine and tyrosine requirements. The organisms surveyed for the presence of these enzymes are set out in Table II. In some cells there is more than one particular enzymatic activity which may, or may not, be associated with a protein complex also carrying out a subsequent reaction (see Fig. 1). ttR. G. H. Cotton and F. Gibson, Biochim. Biophys. dcta 156, 187 (1968). 12See article [70a] for a description of the preparation of prephenate, and article [70b l for an alternative assay for prephenate dehydrogenase.
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AROMATIC AMINO ACIDS
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TABLE II OCCURRENCE OF ENZYMES METABOLIZING GHORISMATE OR PREPHENATE Enzymatic activity present Organism
Chorismate mutase
Prephenate dehydrogenase
Prephenate dehydratase
Reference
Bacteria Aerobacter aerogenes Bacillus subtilis Escherichia coli Pseudomonas aeruginosa Rhodospirillum rubrum Streptococcus cremoris
+ + + + + +
+ + + +
+ + + +
a,d b,c d,e f,g g g
+ + +
+
+
+
+
h i j
+ + --*
+
Fungi Claviceps paspali Neurospora crassa Saccharomyces cerevisiae
Plants Phaseolus aureus Phaseolus vulgaris Pisum savitum
Mammalian cells Ox liver homogenate HeLa cells homogenate
+ +
--*
-
*Not detected, but probably present in cells. ~F. Gibson and J. Pittard, Bacte~4ol. Rev. 32,465 (1968). bE. W. Nester and R. A. Jensen,J. Bacteriol. 91, 1594 (1966). cj. H. Lorence and E. W. Nester, Biochemistry 6, 1541 (1967). aR. G. H. Cotton and F. Gibson, Biochim. Biophys. Acta 100, 76 (1965). ej. Pittard and B.J. Wallace,J. Bacteriol. 91, 1494 (1966). sp. Cerutti, and G. Guroff, J. Biol. Chem. 240, 3034 (1965). °R. G. H. Cotton, unpublished results, 1966. nF. Lingens, W. Goebel, and H. Uesseler, Naturwissenschaften 54, 141 (1967). aT. I. Baker, Biochemistry 5, 2654 (1966). ~F. Lingens, W. Goebel, and H. Uesseler, EuropeanJ. Biochem. 1,363 (1967). ~R. G. H. Cotton and F. Gibson, Biochim. Biophys. Acta 156, 187 (1968). IO. L. Gamborg, and F. J. Simpson, Can.J. Biochem. 42,583 (1964). toO. L. Gamborg, and E. W. Keeley, Biochim. Biophys. Acta 115, 65 (1966).
k,l m k
g g
AROMATIC AMINO ACIDS
[71 ]
[71] Tyrosine and Phenylalanine Biosynthesis: The T and P Proteins (Aerobacter aerogenes); Chorismate Mutase (Pisum sativum)
By R.
G. H. COTTON and F. GIBSON Introduction
The terminal reactions in the biosynthesis of tyrosine and phenylalanine involve the conversion of chorismate through prephenate to 4-hydroxyphenylpyruvate or phenylpyruvate, which are then transaminated to give the amino acids. 1 Although the intermediates are the Phenylpyruvate
~ Phenylalanine
/2
(prephenate)
/1
Chorismate
\3
(prephenate)
\,
4- Hydroxyphenylpyruvate
-'-- Tyrosine
(a)
Phenylpyruvate ~
Phenylalanine
/ Chorismate - -
Prephenate
\
4- Hydroxyphenylpyruvate
~
Tyrosine
(b) FIG. 1. Pathways for the conversion of chorismate to phenylalanine and tyrosine (see text footnote 1). (a) T h e pathways in Aerobacter aerogenes and Escherichia coll. T h e P protein carries out reaction 1 (chorismate mutase P activity) and reaction 2 (prephenate dehydratase activity). T h e T protein carries out reaction 3 (chorismate mutase T activity) and reaction 4 (prephenate dehydrogenase activity). (b) T h e pathways in a n u m b e r of other organisms, e.g., Neurospora and Pisum sativum (the pea). IF. Gibson and .l. Pittard, Bacteriol. Rev. 32,465 (1968).
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TYROSINE AND PHENYLALANINE BIOSYNTHESIS
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same in different types of cells, details of the pathways vary. In Aerobacter aerogenes and in Escherichia coli the conversion of chorismate to 4hydroxyphenylpyruvate is carried out by a single protein or protein complex. 2-4 There is evidence that conversion of chorismate to phenylpyruvate is also carried out by a single protein. 2 These pathways are shown in Fig. la. In some other organisms ~ (see also Table II) only one chorismate mutase activity is present and the pathways branch at prephenate (Fig. 1b).
T Protein Assay Methods Principle. Two reactions are carried out by the T protein: COOH HOOC
/CH2--C--COOH
chorismate mutase
OH
COOH
0
OH
Chorismic acid
Prephenic acid + DPN~prephenat e
~///
dehydrogenase
CH2--C--COOH o + DPNH + H÷ + CO2 OH
O f the two activities carried out by the T protein, chorismate mutase is more readily assayed in crude cell extracts. 5 Chorismate mutase P (see later) is also present in crude cell extracts. Chorismate mutase activity is assayed by estimating the prephenate formed. Prephenate dehydrogenase activity is measured by a fluorometric determination of the DPNH formed during the conversion of prephenate into 4-hydroxyphenylpyruvate. 2R. G. H. Cotton and F. Gibson, Biochim. Biophys..4eta 100, 76 (1965). nR. G. H. Cotton and F. Gibson, Biochim. Biophys. Acta 147,222 (1967). +R. G. H. Cotton and F. Gibson, Biochim. Biophys. Acta 160, 188 (1968). °Prephenate dehydrogenase may be assayed in crude extracts by a modified phenol assay as described by I. Schwinck and E. Adams, Biochim. Biophys. Acta 36, 102 (1959).
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AROMATICAMINOACIDS
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Reagents Chorismic acid, 0.01 M Potassium prephenate, 0.01 M Dithiothreitol, 0.01 M EDTA, 0.001 M Tris-HCl buffer, pH 8.2, 0.5 M DPN +, 0.05 M HC1, M NaOH, M Enzyme
Procedure for Chorismate Mutase Assay Step 1. The incubation mixture contains in a final volume of 1.0 ml: 0.1 ml of chorismic acid, 0.1 ml of dithiothreitol, 0.1 ml of EDTA, 0.1 ml of Tris-HC1 buffer, and about 3 units of enzyme. After incubation for 20 minutes at 37 °, the reaction is stopped by plunging the tubes into an ice bath. Step 2. Prephenate is measured in the incubated samples and in a zero time control by estimating phenylpyruvate formed from prephenate by acid treatment of the samples. From each tube two 0.4-ml samples (A and B) are taken. To A is added 0.4 ml of HCI arid tke mixture incubated 10 minutes at 37 ° after which 3.2 ml of NaOH is added and the absorbance at 320 m/x is measured. To B is added 3.2 ml of N a O H followed by 0.4 ml of HCI, and the absorbance at 320 m/x is measured. The difference in absorbance at 320 m/z between A and B indicates the amount of phenylpyruvate formed from prephenate. Phenylpyruvate has a molar absorption coefficient of 17,500 in alkaline solution. The absorbance is measured as soon as possible after the addition of alkali, as the chromophore is unstable, n In each experiment a control without enzyme is included to estimate the small amount of prephenate formed nonenzymatically from chorismate during the incubation period. Chorismate mutase may also be assayed by following the disappearance of chorismate spectrophotometrically? Definition of Unit and Specific Activity. One unit of enzyme activity is defined as the amount catalyzing the formation of 0.1 micromole of prephenate in 20 minutes at 37 °. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. T eC. H. Doy and F. Gibson, Biochim. Biophys. Acta 50, 495 (1961). 70. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951); see also Vol. III [73].
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TYROSINE AND PHENYLALANINE BIOSYNTHESIS
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Procedure for Prephenate Dehydrogenase Assay The incubation mixture contains in a final volume of 1.5 ml in a quartz cuvette: 0.075 ml of potassium prephenate., 0.075 ml of Tris-HC1 buffer, 0.03 ml of DPN +, 0.15 ml of dithiothreitol, 0.15 ml of EDTA, and about 3 units of enzyme. The reaction mixture without DPN + is incubated for 3 minutes in a 37 ° water bath and then transferred to a temperature-controlled (37 °) compartment of an Aminco-Bowman, or similar spectrophotofluorometer. The reaction is started by the addition of DPN +, and the initial rate of increase of fluorescence (340 m/~ activation, 440 m/~ fluorescence; uncorrected) is measured on an X-Y recorder for about 4 minutes. The concentration of DPNH formed is measured using a standard curve prepared by measuring the fluorescence of known concentrations of DPNH. Purified preparations of prephenate dehydrogenase may also be measured spectro photometrically. 3 Definition of Unit and Specific Activity. One unit of enzyme activity is taken as that amount catalyzing the formation of 0.1 micromole of DPNH in 20 minutes at 37 °. Specific activity is expressed in units per milligram of protein. Protein is determined by the procedure of Lowry
et al.7 Purification Procedure A multiple aromatic auxotroph of .4. aerogenes (strain poly 3 was used in the purification described) TM is grown in a medium in which growth is limited by tyrosine, thus derepressing the T protein about 3-fold with respect to the wild-type strain grown in minimal medium. Several buffers are used: Buffer A: 0.01 M potassium phosphate (pH 7.0) containing 10 -3 M mercaptoethanol and 10 -4 M EDTA. Buffer B: as for buffer A, except pH 6.0 Buffer C: 0.025 M potassium phosphate (pH 8.0) containing 10-3 M mercaptoethanol and 10-4 M EDTA Buffer D: 0.1 M citric acid-potassium phosphate (pH 3.8) Buffer E: 0.2 M potassium phosphate (pH 8.0) containing 10-a M mercaptoethanol, 10-4 M EDTA and 0.3 M sodium chloride Buffer F: as for buffer A, but including 0.05 M sodium chloride
Step 1. Growth of Cells and Preparation of Cell-Free Extract. Twenty nutrient agar slopes of.4. aerogenes poly 3 are grown overnight at 37 ° and ~aAerobacter aerogenes poly 3 may be obtained from F. Gibson, Biochemistry Department,
I.A.S. Australian National University, Canberra, A.C.T., Australia.
568
AROMATIC AMINOACIDS
[71 ]
the growth is washed off with sterile glucose solution as inoculum for 40 liters of culture medium (glucose (0.16%)-citrate-mineral salts medium s containing 10-4 M L-tryptophan, 2 × 10-4 M L-phenylalanine, 10-n M p-aminobenzoic acid, and 9 × 10-5 M L-tyrosine). The culture is stirred and aerated at 37 ° until 1 hour after growth ceases. T h e absorbance of the fully grown culture is 1.5 at 600 m/~ in a 1-cm cell. The culture is stored at 0-4 ° overnight, and the cells are harvested by centrifugation. The cells are then washed by suspending them in about five times their packed volume of 0.9% NaCI, then centrifuging at 4 ° for 15 minutes at 10,000 g. T h e cells are then suspended in 4 ml of buffer A for each gram wet weight and smashed in a Ribi cell disintegrator at 20,000 psi (or French press). Whole cells and cell debris are removed by centrifugation at 17,000 g for 20 minutes. The supernatant (about 200 ml) is decanted and stored at--20 °. All the subsequent steps are carried out at 0-4 °. Step 2. Removal of Nucleic Acid. To each 200 ml of crude extract is added 33.3 ml of neutralized 0.4% protamine sulfate, and the mixture is stirred for 20 minutes. After centrifugation at 17,000 g for 20 minutes, the supernatant is decanted. Step 3. Treatment at pH 5.5. An equal volume of cold buffer D is added rapidly to the supernatant from step 2, and the mixture is stirred for 70 minutes. The final pH is about 5.5. After centrifugation at t2,000 g for 20 minutes, the supernatant is discarded and the precipitate is resuspended in an equal volume of buffer E and stirred for 1 hour. The supernatant obtained after a further centrifugation at 12,000 g for 20 minutes is used for the next step. Step 4. Ammonium Sulfate Treatment. Solid ammonium sulfate is added over a period of 15 minutes to give 40% saturation, and the solution stirred for a further 30 minutes. The precipitate which is sedimented at 12,000 g for 20 minutes is suspended in the minimum volume of buffer F and then dialyzed twice against 2 liters of buffer F for 1 hour. Step 5. Sephadex Chromatography. The dialyzed solution (up to 40 ml) is then applied to a 4.2 × 115 cm column of Sephadex G-100 and eluted with buffer F. The active fractions are pooled. Step 6. DEAE-Cellulose Chromatography. The pooled active fractions are applied to a 2.6 x 20 cm DEAE-cellulose column equilibrated with buffer A. The sample is washed in with 20 ml of buffer A containing 0.1 M NaC1. The column is then treated by linear gradient etution starting with 500 ml of buffer A containing 0.1 M NaC1 in the mixing bottle and 500 ml of the same buffer containing 0.2 M NaCI in the inlet SH. J. Vogel, and D. M. Bonner,J. Biol. Chem. 218, 97 (1956); see also this volume [ 1 ].
[71]
TYROSINE AND PHENYLALANINE BIOSYNTHESIS
569
bottle. Elution is completed by additional buffer A containing 0.2 M NaCI. Fractions are collected every 16 minutes with a flow rate of 75 ml per hour. Chorismate mutase P appears at the front and is discarded, whereas the T protein appears in fractions 36-56. Step 7. Concentration by DEAE-Cellulose Chromatography. The active fractions (other than chorismate mutase P) are pooled and diluted with an equal volume of buffer A; the enzyme is concentrated on two 2 x 13 cm DEAE-cellulose columns equilibrated with buffer A. Half of the enzyme solution is applied to each of the columns at about 250 ml per hour, and then the adsorbed protein is quickly eluted with buffer E. The active fractions at the protein front are pooled and dialyzed three times for 1 hour against 2 liters of buffer B. Step 8. Chromatography on Brushite. The dialyzed sample is applied to a 2.6 x 20 cm column of freshly decanted Brushite 9 equilibrated with buffer B. After washing in the sample with buffer B, a linear gradient is applied starting with 400 ml of buffer B in the mixing flask and 400 ml of buffer C in the inlet flask. The protein is eluted with buffer at 75 mi per hour; fractions are collected at 16-minute intervals and the elution is completed with further buffer C. The T protein appears in fractions 46-64. Step 9. Concentration by DEAE-Cellulose Chromatography. The active fractions are pooled and applied to a 1.5 x 10 cm DEAE-cellulose column at 200 ml per hour. Protein is then quickly eluted with buffer E most of the protein being collected in less than 5 ml. A summary of the purification is given in Table I. The loss of activity following elution from Brushite and its subsequent recovery following concentration on DEAE-cellulose are probably due to the reversible formation of inactive subunits? ,4
Properties Stability. Concentrated solutions of the enzyme appear to be stable on storage at - 2 0 °. About 5% loss of activity is observed on freezing and thawing. In dilute solutions at 4 °, activity is lost but may be largely recovered by concentration. Dialysis and freezing causes inactivation which is reversed by thiols. Purity. Polyacrylamide gel disc electrophoresis reveals one major band of protein, with several slower moving, and one faster moving, faint bands. The heavy band can be shown to correspond to the prephenate dehydrogenase activity using a dehydrogenase stain technique? ° 9A. Tiselius, S. Hjert6n, and O. Levin, Arch. Biochem. Biophys. 65, 132 (1956); see also Vol.
v[2]. 1°C. L. Markert and F. M~ller, Proc. Natl. Acad. Sci. U.S. 45,753 (1959).
570
AROMATIC AMINO ACIDS
[71]
TABLE I SUMMARYOF PURIFICATION PROCEDUREOF Y PROTEINa
Protein
Step
Fraction
1 2 3 4
Cell-free extract Protamine sulfate supernatant pH precipitate 0-40% Ammonium sulfate
200 200 196 30
578 392 201 1035
5 6 7
Sephadex chromatography DEAE-cellulosechromatography DEAE-cellulosechromatography (concentration) Brushite DEAE-cellulosechromatography
150 415 45
164 33 256 3.7 1037
8 9
Specific activity (units/mg
Volume Units/ml (mg/ml) protein)
320 2.5
22.6 12.4 3.9 14.4
Yield
(%)
26 32 52 72
100 68 34 27
0.62 0.07 0.41
265 470 625
21 12 10
0.012 0.38
310 2720
1 2.3
aThe purification is followed by measuring chorismate mutase activity; therefore values
for specific activity given for steps 1-4 are approximate because of the presence of some P protein (about 30% of the chorismate mutase activity in the crude cell extract), which is finallycompletely separated during step 6. Kinetic Properties of the T Protein. 3 Chorismate mutase T has a p H o p t i m u m at about 8.5 in Tris-HC1 and p r e p h e n a t e d e h y d r o g e n a s e has highest activity at p H values above 8.6. Reciprocal plots o f rate-concentration p a r a m e t e r s for chorismate mutase are usually n o n l i n e a r at low concentrations o f chorismate. At high concentrations the plots are linear, giving an a p p a r e n t Km for chorismate o f 5.6 × 10 -4 M. For p r e p h e n a t e d e h y d r o g e n a s e the app a r e n t Km for DPN is 1.5 × 10 -4 M and for p r e p h e n a t e 1.1 × 10 -4 M. T P N has about half the activity o f DPN as a cofactor. Inhibitors and Activators. 3 T h e e n d - p r o d u c t inhibitor, L-tyrosine at 10 -4 M, inhibits p r e p h e n a t e d e h y d r o g e n a s e 50%, but does not affect chorismate mutase T. P r e p h e n a t e d e h y d r o g e n a s e was inhibited 50% by 4 - h y d r o x y c i n n a m a t e , 4 - h y d r o x y p h e n y l p y r u v a t e , a n d 4 - h y d r o x y phenyllactate, each at 10 -3 M, a n d by D-tyrosine and 4 - h y d r o x y b e n z o a t e , each at 10 -2 M. T y r a m i n e is not an inhibitor. S o d i u m dodecyl sulfate or cupric sulfate inhibited 50% at 10 -5 M a n d 10 -4 M, respectively. Only the last two c o m p o u n d s affect chorismate mutase T, reversibly inactivating the enzyme. T h e effects o f inhibitors o n the kinetics have been discussed? Antibody against the T protein inhibits chorismate mutase activity u p to 95%. Molecular Weight and Subunit Structure. 4 T h e T protein has a molecular weight o f about 87,000 but is dissociable into subunits without choris-
[71 ]
TYROSINE AND PHENYLALAN1NEBIOSYNTHESIS
571
mate mutase or prephenate dehydrogenase activity by treatment with either acid or sodium dodecyl sulfate. The subunits, which have a molecular weight of about 45,000, can be recombined to give active protein carrying out both of the reactions.
P Protein The P protein has not been extensively purified; a partial purification, on a small scale, together with some properties of the protein is described.
Assay Methods
Principle. The P protein has two enzymatic activities, namely, chorismate mutase (1) and prephenate dehydratase (2). Chorismate Prephenate
, prephenate
(1)
~ phenylpyruvate+ H20 + CO2
(2)
Chorismate mutase is assayed as described for the T protein. Prephenate dehydratase is assayed by estimating the phenylpyruvate formed from prephenate.
Reagents Potassium prephenate, 0.01 M Tris-HCl buffer, 0.25 M, pH 8.2 NaOH, M Enzyme
Procedure. The incubation mixture contains, in a final volume of 1 ml: 0.05 ml of potassium prephenate, 0.1 ml of Tris-HCl buffer, and about 3 units of enzyme. After incubation for 30 minutes at 37 °, 3.0 ml of N a O H is added and the absorbancy at 320 m/x is measured. Phenylpyruvate is estimated using a molar absorption coefficient of 17,500. An unincubated control is also assayed for phenylpyruvate. Definition of Unit and Specific Activity. One unit of enzyme activity is defined as the amount catalyzing the formation of 0.1 micromole of phenylpyruvate in 20 minutes at 37 °. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al/ Purification Procedure The partial purification of the P protein from a wild-type strain of
A. aerogenes (NCW) is described. The same method has also been used
572
AROMATIC AMINO ACIDS
[71]
with the P protein from E. coil W and E. coil K12, as wall as another strain of A. aerogenes. Step 1. Preparation of Cell-Free Extract. Cells are grown in a citratemineral salts medium 8 supplemented with 0.16% glucose. One-liter quantities of medium are inoculated with 1 ml from a 5-hour nutrient broth culture and incubated with shaking at 37 °. Cells are harvested in the late logarithmic phase and collected by centrifugation at 12,000 g for 20 minutes. The cells are washed in 0.9% NaC1 (one-twelfth the original medium volume) and finally suspended in 4 ml of 0.1 M potassium phosphate buffer (pH 7.0) for each gram wet weight of cells. After disintegration in a French pressure cell at 20,000 psi, the unbroken cells and cell debris are removed by centrifugation at 17,000 g for 15 minutes. About 4 ml of cell-free extract is obtained from each liter of culture. The crude cell-free extract is stored at --20 °. Step 2. Chromatography on DEAE-Cellulose. The cell-free extract is dialyzed twice for 1 hour against 2 liters of 0.01 M potassium phosphate buffer (pH 7.0) (the column buffer), and 4 ml is applied to a 2 × 20 cm column of DEAE-cellulose previously equilibrated against the above buffer. After washing in the protein with 2 lots of 5 ml of the column buffer, the column is then treated by linear gradient elution with 400 ml of the column buffer in the mixing flask and 400 ml of the same buffer containing 0.5 M NaCI in the inlet bottle. The eluent is passed through the column at 150 ml per hour, and 7.5-ml fractions are collected. The P protein (chorismate mutase P and prephenate dehydratase activities) is eluted about fraction 33 and the T protein at about fraction 58. A'. second unstable prephenate dehydratase (A) is present in A. aerogenes NCW and is eluted at the protein front. Properties ~
Stability. The P protein from A. aerogenes is unstable at 4 ° and is protected against heat by phenylalanine. The protein from E. coli is more stable. Inhibitors. Prephenate dehydratase is completely inhibited by 10 -3 M L-phenylalanine and inhibited 4 4 % by 5..× 10 -3 M L-tyrosine. Chorismate mutase P is inhibited 6 5 % by 5 × 10 ~3 M I:phenyialanine and 16% by 5 × 10-3 M l:tyrosine. Prephenate dehydratase A is relatively unaffected by the amino acids. Chorismate mutase P is inhibited by high concent~rat4ons o f thiol reagents and also by ferrous ions and detergent. Chorismate Mutase from Peas (Pisum sativum) There appears to be only a single chorismate mutase 4n the pea
[71]
TYROSINE AND PHENYLALANINE BIOSYNTHESIS
573
where the steps correspond to those shown in Fig. lb. The enzyme has been examined after chromatography on DEAE-cellulose n as described below. Assays are carried out as previously described for P protein. Step 1. Preparation of CeU-Free Extract. Seedlings are grown for 10 days in moist sterile absorbent cotton wool. A 10-g quantity of cotyledons is homogenized in a Waring blendor for 1 minute with 10 ml. of 0.1 M Tris-HCl buffer (pH 7.8) containing 0.025 M sodium thioglycolate. The extract is then filtered through gauze and centrifuged at 17,000 g for 20 minutes. After dialysis twice for 1 hour against 0.01 M potassium phosphate buffer (pH 7.0), the extract is centrifuged again; the supernatant is used for the next step. Step 2. Chromatography on DEAE-Cellulose. The enzyme solution is chromatographed on a 2 × 30 cm DEAE-cellulose column previously equilibrated with 0.01 M phosphate buffer (pH 7.0) (column buffer). The column is then treated with a linear gradient starting with 500 ml of the column buffer in the mixing flask and 500 ml of the same buffer containing 0.5 M NaCI in the inlet flask. The flow rate is 150 ml per hour, 10-ml fractions being collected every 4 minutes. Activity appears in fraction 39. Properties t 1
Inhibitors and Activators. DL-Tryptophan (1.2 X 10-4 M) stimulated the activity in crude cell extracts about 20% and in column fractions up to 3-fold. This concentration of tryptophan was included in all assays. L-Phenylalanine and L-tyrosine both inhibit the enzyme, their effects being antagonized by DL-tryptophan. Related Enzymes. Prephenate dehydrogenase or prephenate dehydratase were not detectable in extracts of pea cotyledons prepared as described. TM Distribution o f Enzymatic Activities Related to Chorismate and Prephenate Metabolism
The enzymes described are widely distributed, probably being present in all organisms which synthesize their own phenylalanine and tyrosine requirements. The organisms surveyed for the presence of these enzymes are set out in Table II. In some cells there is more than one particular enzymatic activity which may, or may not, be associated with a protein complex also carrying out a subsequent reaction (see Fig. 1). ttR. G. H. Cotton and F. Gibson, Biochim. Biophys. dcta 156, 187 (1968). 12See article [70a] for a description of the preparation of prephenate, and article [70b l for an alternative assay for prephenate dehydrogenase.
574
AROMATIC AMINO ACIDS
[71]
TABLE II OCCURRENCE OF ENZYMES METABOLIZING GHORISMATE OR PREPHENATE Enzymatic activity present Organism
Chorismate mutase
Prephenate dehydrogenase
Prephenate dehydratase
Reference
Bacteria Aerobacter aerogenes Bacillus subtilis Escherichia coli Pseudomonas aeruginosa Rhodospirillum rubrum Streptococcus cremoris
+ + + + + +
+ + + +
+ + + +
a,d b,c d,e f,g g g
+ + +
+
+
+
+
h i j
+ + --*
+
Fungi Claviceps paspali Neurospora crassa Saccharomyces cerevisiae
Plants Phaseolus aureus Phaseolus vulgaris Pisum savitum
Mammalian cells Ox liver homogenate HeLa cells homogenate
+ +
--*
-
*Not detected, but probably present in cells. ~F. Gibson and J. Pittard, Bacte~4ol. Rev. 32,465 (1968). bE. W. Nester and R. A. Jensen,J. Bacteriol. 91, 1594 (1966). cj. H. Lorence and E. W. Nester, Biochemistry 6, 1541 (1967). aR. G. H. Cotton and F. Gibson, Biochim. Biophys. Acta 100, 76 (1965). ej. Pittard and B.J. Wallace,J. Bacteriol. 91, 1494 (1966). sp. Cerutti, and G. Guroff, J. Biol. Chem. 240, 3034 (1965). °R. G. H. Cotton, unpublished results, 1966. nF. Lingens, W. Goebel, and H. Uesseler, Naturwissenschaften 54, 141 (1967). aT. I. Baker, Biochemistry 5, 2654 (1966). ~F. Lingens, W. Goebel, and H. Uesseler, EuropeanJ. Biochem. 1,363 (1967). ~R. G. H. Cotton and F. Gibson, Biochim. Biophys. Acta 156, 187 (1968). IO. L. Gamborg, and F. J. Simpson, Can.J. Biochem. 42,583 (1964). toO. L. Gamborg, and E. W. Keeley, Biochim. Biophys. Acta 115, 65 (1966).
k,l m k
g g