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The biosynthesis of phenylalanine and tyrosine in the pea (Pisum sativum): Chorismate mutase
BIOCHIMICA ET BIOPHYSICA ACTA
SHORT
187
COMMUNICATIONS
BBA 2338O
The biosynthesis of phenylalanine and tyrosine in the pea (P/sum sativum): Chorismate mutase The terminal reactions in the biosynthesis of tyrosine and phenylalanine have been well established for Escherichia coli and Aerobacter aerogenes1-3. In these organisms chorismate is converted by two proteins (T and P proteins) through prephenate towards tyrosine and phenylalanine, respectively (Fig. I).
I
>
~
7 p - h y d r o x y p h e n y l p y r u v a t e --+ tyrosine
Chorismate _ _ _ ~ prephenate 2
"\
4~ p h e n y l p y r u v a t e --+ phenylalanine Fig. I. The terminal reactions involved in the biosynthesis of phenylalanine and tyrosine in A. aerogenes and E. coll. The enzymes are: i, chorismate m u t a s e T; 2, chorismate m u t a s e P; 3, prephenate dehydrogenase; 4, prephenate dehydratase. E n z y m e activities I and 3 are possessed by the T protein and 2 and 4 by the P protein.
Several studies have indicated that the pathways of synthesis of phenylalanine and tyrosine in plants m a y involve the same intermediates as in bacterial cells 44, but chorismate mutase activity has not actually been demonstrated. Prephenate dehydrogenase has been demonstrated in mung bean (Phaseolus aureus) as have the transaminases capable of forming phenylalanine and tyrosine from the corresponding keto acids 9. Evidence for the presence of prephenate dehydratase in this plant has also been obtained ~ and prephenate dehydrogenase has been purified from wax bean and found insensitive to end-product inhibition by tyrosine 1°. Chorismate mutase activity has now been demonstrated in peas (Pisum satiw~m) and also in the mung bean. The effect of aromatic amino acids on chorismate mutase activity in the pea has been examined and the enzyme partially purified b y chromatography. Seedlings were grown for IO days on sterile absorbent cotton wool (moistened as required) after which io g of cotyledons were homogenised in a vitamiser for I min with Io ml of o.I M Tris-HC1 buffer (pH 7.8) containing 0.025 M sodium thioglycolate. After filtration through gauze the cell extract was centrifuged (16500 × g for 20 min) before and after dialysis for I h against 2 lots of 2 1 of o.oi M potassium phosphate buffer (pH 7.0). Chorismate mutase was assayed b y estimating the prephenate formed from chorismate by conversion to phenylpyruvate as described previously 2. Chorismate mutase was inhibited b y either phenylalanine or tyrosine and the inhibitions were not additive when both amino acids were added to the reaction mixture (Table I). Crude cell extract was chromatographed on DEAE-cellulose columns (2 cm Biochim. Biophys. Acta, 156 (I968) 187-189
188
SHORT COMMUNICATIONS
TABLE THE
I
INHIBITION
OF
CHORISMATI~
MUTASE
BY
PHENYLALAN1NE
AND
TYROSINE
T h e r e a c t i o n m i x t u r e w a s i n c u b a t e d f o r 2 0 r a i n a t 37 ° a n d c o n t a i n e d i n a t o t a l v o l u m e of I m l : 5 ° / ~ m o l e s T r i s - H C 1 b u f f e r ( p H 8.2), I , u m o l e E D T A , 1.5 t * m o l e s c h o r i s m i c a c i d a n d 0. 3 m l (6.6 m g p r o t e i n ) of a c r u d e e x t r a c t of p e a c o t y l e d o n s . I n h i b i t i o n is e x p r e s s e d a s t h e p e r c e n t a g e of a n u n i n h i b i t e d c o n t r o l w h i c h f o r m e d 0 . 3 o # m o l e of p r e p h e n a t e p e r m l of r e a c t i o n m i x t u r e .
Addition
Concn, ,'< lO 3 (M)
L-Phenylalanine
I 4 I 4 both i
L-Tyrosine L - P h e n y l a l a n i n e plus L - t y r o s i n e
fnhibition (% ) 4° 64 5° 69 5°
× 30 cm) as previously described for cell extracts o f A . aerogenes 2 except that there was 5oo ml of buffer in each bottle for the gradient elution and the io-ml fractions were collected each 4 rain. Only a single peak of low activity was detected containing 17 % of the activity applied to the column. This low activity was not inhibited by phenylalanine and tyrosine. Subsequently it was found that low levels of tryptophan (1.2" lO-4 M) activated chorismate mutase from the columns up to 3-fold. The activity in the crude extract was only stimulated about 2o % b y this concentration of tryptophan. Activated enzyme was inhibited by phenylalanine or tyrosine and it was found that the higher levels of tryptophan reversed their inhibition (Table II). TABLE
II
ACTIVATION OF CHORISMATE MUTASE BY TRYPTOPHAN AND REVERSAL OF END-PRODUCT INHIBITION D i a l y s e d e n z y m e p r e p a r a t i o n (see t e x t ) w a s p a r t i a l l y p u r i f i e d b y b a t c h e l u t i o n o f i o m l of a c r u d e e x t r a c t of p e a c o t y l e d o n s f r o m a 2 c m × 3 ° c m D E A E - c e l l u l o s e column. Unadsorbed m a t e r i a l w a s w a s h e d t h r o u g h w i t h o . o i M p h o s p h a t e b u f f e r ( p H 7.0) a n d e n z y m e a c t i v i t y w a s e l u t e d w i t h t h i s b u f f e r c o n t a i n i n g 0.2 M NaC1. C h o r i s m a t e m u t a s e a c t i v i t y w a s m e a s u r e d a s d e s c r i b e d in T a b l e I.
Addition to reaction mixture (M) L-Phenylalanine L-Tyrosine
DL-Tryptophan
--
--
- -
- -
1.2.
lO
-4
10 -3
--
--
1,2"
IO -3 i o -a
---
1.2"IO 4 1.2 • 1o -a
--
10
--
I0 -3
-3
Prephenate formed (#moles/ml of reaction mixture) 0.09 o.17 O.18
0.08 o. i 5
1.2"
10 -4
0.I0
1.2"
IO -3
0.I 7
Attempts to demonstrate the presence of prephenate dehydratase and prephenate dehydrogenase in the crude cell extracts using methods previously described 2 were not conclusive. It is possible that the products were further metabolised as it was noted that prephenate disappeared and that the small amount of phenylpyruvate present in the prephenate solution was removed during incubation. Biochim. Biophys. Acta, i 5 6 ( i 9 6 8 ) i 8 7 - i 8 9
SHORT COMMUNICATIONS
189
There are similarities between the chorismate mutase activities of Neurospora and Saccharomyces and the activity in P. sativum. Thus tryptophan has been shown to activate chorismate mutase and reverse end-product inhibitionn, ~2 (T. I. BAKER, private communication) in cell-free preparations from the former organisms. Single peaks of chorismate mutase activity following DEAE-cellulose chromatography have also been found in extracts from Neurospora (T. I. BAKER, private communication), Pseudornonas aeruginosa~4 and Claviceps ~3. Chorismate mutase and related enzymes have been studied in other organisms 15-1s and there is variation in the number of chorismate mutase activities and effects of end products. Further examination of the biosynthetic pathways leading to phenylalanine and tyrosine may be profitable in the study of evolutionary relationships. The Australian National Health and Medical Research Council, the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health (Grant AM o4632) and the University of Melbourne are thanked for research grants.
School of Microbiology, University of Melbourne, Parkville, Victoria (Australia)
R, G. H. COTTON* F . GIBSON*
I A. MEISTER, Biochemistry of the Amino Acids, Academic Press, London, 2nd ed., 1965, p. 2o 4. 2 R. G. H. COTTON ANO F. GIBSON, Biochim. Biophys. Acta, IOO (1965) 76. 3 J. PITTARD AND B. J. "WALLACE, J. Bacteriol., 91 (1966) 1494. 4 M. NANO'x" ANON. C. GANGULI, .4rch. Biochem. Biophys., 92 (1961) 399. 5 M. NANDY AND N. C. GANGULI, Biochim. Biophys. Acta, 48 (1961) 608. 6 D. BALINSKY AND D. D. DAVIES, Biochem. J., 80 (1961) 300. 7 D. BALINSI
Received September I8th, 1967 * Present address: D e p a r t m e n t of Biochemistry, J o h n Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 26oo, Australia.
Biochim. Biophys. Acta, 156 (1968) 187-189
SHORT
187
COMMUNICATIONS
BBA 2338O
The biosynthesis of phenylalanine and tyrosine in the pea (P/sum sativum): Chorismate mutase The terminal reactions in the biosynthesis of tyrosine and phenylalanine have been well established for Escherichia coli and Aerobacter aerogenes1-3. In these organisms chorismate is converted by two proteins (T and P proteins) through prephenate towards tyrosine and phenylalanine, respectively (Fig. I).
I
>
~
7 p - h y d r o x y p h e n y l p y r u v a t e --+ tyrosine
Chorismate _ _ _ ~ prephenate 2
"\
4~ p h e n y l p y r u v a t e --+ phenylalanine Fig. I. The terminal reactions involved in the biosynthesis of phenylalanine and tyrosine in A. aerogenes and E. coll. The enzymes are: i, chorismate m u t a s e T; 2, chorismate m u t a s e P; 3, prephenate dehydrogenase; 4, prephenate dehydratase. E n z y m e activities I and 3 are possessed by the T protein and 2 and 4 by the P protein.
Several studies have indicated that the pathways of synthesis of phenylalanine and tyrosine in plants m a y involve the same intermediates as in bacterial cells 44, but chorismate mutase activity has not actually been demonstrated. Prephenate dehydrogenase has been demonstrated in mung bean (Phaseolus aureus) as have the transaminases capable of forming phenylalanine and tyrosine from the corresponding keto acids 9. Evidence for the presence of prephenate dehydratase in this plant has also been obtained ~ and prephenate dehydrogenase has been purified from wax bean and found insensitive to end-product inhibition by tyrosine 1°. Chorismate mutase activity has now been demonstrated in peas (Pisum satiw~m) and also in the mung bean. The effect of aromatic amino acids on chorismate mutase activity in the pea has been examined and the enzyme partially purified b y chromatography. Seedlings were grown for IO days on sterile absorbent cotton wool (moistened as required) after which io g of cotyledons were homogenised in a vitamiser for I min with Io ml of o.I M Tris-HC1 buffer (pH 7.8) containing 0.025 M sodium thioglycolate. After filtration through gauze the cell extract was centrifuged (16500 × g for 20 min) before and after dialysis for I h against 2 lots of 2 1 of o.oi M potassium phosphate buffer (pH 7.0). Chorismate mutase was assayed b y estimating the prephenate formed from chorismate by conversion to phenylpyruvate as described previously 2. Chorismate mutase was inhibited b y either phenylalanine or tyrosine and the inhibitions were not additive when both amino acids were added to the reaction mixture (Table I). Crude cell extract was chromatographed on DEAE-cellulose columns (2 cm Biochim. Biophys. Acta, 156 (I968) 187-189
188
SHORT COMMUNICATIONS
TABLE THE
I
INHIBITION
OF
CHORISMATI~
MUTASE
BY
PHENYLALAN1NE
AND
TYROSINE
T h e r e a c t i o n m i x t u r e w a s i n c u b a t e d f o r 2 0 r a i n a t 37 ° a n d c o n t a i n e d i n a t o t a l v o l u m e of I m l : 5 ° / ~ m o l e s T r i s - H C 1 b u f f e r ( p H 8.2), I , u m o l e E D T A , 1.5 t * m o l e s c h o r i s m i c a c i d a n d 0. 3 m l (6.6 m g p r o t e i n ) of a c r u d e e x t r a c t of p e a c o t y l e d o n s . I n h i b i t i o n is e x p r e s s e d a s t h e p e r c e n t a g e of a n u n i n h i b i t e d c o n t r o l w h i c h f o r m e d 0 . 3 o # m o l e of p r e p h e n a t e p e r m l of r e a c t i o n m i x t u r e .
Addition
Concn, ,'< lO 3 (M)
L-Phenylalanine
I 4 I 4 both i
L-Tyrosine L - P h e n y l a l a n i n e plus L - t y r o s i n e
fnhibition (% ) 4° 64 5° 69 5°
× 30 cm) as previously described for cell extracts o f A . aerogenes 2 except that there was 5oo ml of buffer in each bottle for the gradient elution and the io-ml fractions were collected each 4 rain. Only a single peak of low activity was detected containing 17 % of the activity applied to the column. This low activity was not inhibited by phenylalanine and tyrosine. Subsequently it was found that low levels of tryptophan (1.2" lO-4 M) activated chorismate mutase from the columns up to 3-fold. The activity in the crude extract was only stimulated about 2o % b y this concentration of tryptophan. Activated enzyme was inhibited by phenylalanine or tyrosine and it was found that the higher levels of tryptophan reversed their inhibition (Table II). TABLE
II
ACTIVATION OF CHORISMATE MUTASE BY TRYPTOPHAN AND REVERSAL OF END-PRODUCT INHIBITION D i a l y s e d e n z y m e p r e p a r a t i o n (see t e x t ) w a s p a r t i a l l y p u r i f i e d b y b a t c h e l u t i o n o f i o m l of a c r u d e e x t r a c t of p e a c o t y l e d o n s f r o m a 2 c m × 3 ° c m D E A E - c e l l u l o s e column. Unadsorbed m a t e r i a l w a s w a s h e d t h r o u g h w i t h o . o i M p h o s p h a t e b u f f e r ( p H 7.0) a n d e n z y m e a c t i v i t y w a s e l u t e d w i t h t h i s b u f f e r c o n t a i n i n g 0.2 M NaC1. C h o r i s m a t e m u t a s e a c t i v i t y w a s m e a s u r e d a s d e s c r i b e d in T a b l e I.
Addition to reaction mixture (M) L-Phenylalanine L-Tyrosine
DL-Tryptophan
--
--
- -
- -
1.2.
lO
-4
10 -3
--
--
1,2"
IO -3 i o -a
---
1.2"IO 4 1.2 • 1o -a
--
10
--
I0 -3
-3
Prephenate formed (#moles/ml of reaction mixture) 0.09 o.17 O.18
0.08 o. i 5
1.2"
10 -4
0.I0
1.2"
IO -3
0.I 7
Attempts to demonstrate the presence of prephenate dehydratase and prephenate dehydrogenase in the crude cell extracts using methods previously described 2 were not conclusive. It is possible that the products were further metabolised as it was noted that prephenate disappeared and that the small amount of phenylpyruvate present in the prephenate solution was removed during incubation. Biochim. Biophys. Acta, i 5 6 ( i 9 6 8 ) i 8 7 - i 8 9
SHORT COMMUNICATIONS
189
There are similarities between the chorismate mutase activities of Neurospora and Saccharomyces and the activity in P. sativum. Thus tryptophan has been shown to activate chorismate mutase and reverse end-product inhibitionn, ~2 (T. I. BAKER, private communication) in cell-free preparations from the former organisms. Single peaks of chorismate mutase activity following DEAE-cellulose chromatography have also been found in extracts from Neurospora (T. I. BAKER, private communication), Pseudornonas aeruginosa~4 and Claviceps ~3. Chorismate mutase and related enzymes have been studied in other organisms 15-1s and there is variation in the number of chorismate mutase activities and effects of end products. Further examination of the biosynthetic pathways leading to phenylalanine and tyrosine may be profitable in the study of evolutionary relationships. The Australian National Health and Medical Research Council, the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health (Grant AM o4632) and the University of Melbourne are thanked for research grants.
School of Microbiology, University of Melbourne, Parkville, Victoria (Australia)
R, G. H. COTTON* F . GIBSON*
I A. MEISTER, Biochemistry of the Amino Acids, Academic Press, London, 2nd ed., 1965, p. 2o 4. 2 R. G. H. COTTON ANO F. GIBSON, Biochim. Biophys. Acta, IOO (1965) 76. 3 J. PITTARD AND B. J. "WALLACE, J. Bacteriol., 91 (1966) 1494. 4 M. NANO'x" ANON. C. GANGULI, .4rch. Biochem. Biophys., 92 (1961) 399. 5 M. NANDY AND N. C. GANGULI, Biochim. Biophys. Acta, 48 (1961) 608. 6 D. BALINSKY AND D. D. DAVIES, Biochem. J., 80 (1961) 300. 7 D. BALINSI
Received September I8th, 1967 * Present address: D e p a r t m e n t of Biochemistry, J o h n Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 26oo, Australia.
Biochim. Biophys. Acta, 156 (1968) 187-189