- Email: [email protected]
Induced, soluble phenylalanine hydroxylase from Pseudomonas sp. grown on phenylalanine or tyrosine
159
PRELIMINARY NOTES
to be closely r e l a t e d t o - - i f n o t i d e n t i c a l w i t h - - t h e s y s t e m effecting aerobic h y d r o x y l a tion of a l k a n e s to give t h e p r i m a r y alcohol. Koninklijke/Shell Laboratorium, Amsterdam, Shell Internationale Research Maatschappij, N . V . , 's Gravenhage ( T h e Netherlands)
A. C. VAN DER LINDEN
1 G. J. E. THIJSSE AND A. C. VAN DER LINDEN, Antonie van Leeuwenhoek, 29 (z963) 89. E. R. LEADBETTER AND J. W. FOSTER, Arch. Microbiol., 35 (196o) 92. 3 J. E. STEWART, R. E. KALLIO, D. P. STEVENSON, A. C. JONES AND D. O. SCHISSLER, J.Bacteriol., 78 (I959) 441. J. B. DAVIS AND H. F. YARBROUGH, Science, 137 (1962) 615. J. CHOUTEAU, E. AZOULAYAND J. C. SENEZ, Nature, 194 (1962) 576. R. E. PARKER AND INT.S. ISAACS,Chem. Revs., (1959) 7377 B. i . BLOOM AND G. M. SHULL, J. Am. Chem. Soc.. 77 (1955) 5767 . s C. J. SIH, J. Bacteriol., 84 (1962) 382. 9 H. ICHIHARA, t(. ADACHI, K. HOSOKAWAAND Y. TAKEDA, J. Biol. Chem., 237 (1962) 2296. x0 H. S. MASON, Advan. Enzymol., 19 (I957) 79. R e c e i v e d A p r i l 24th, 1963 Biochim. Biophys. Acta, 77 (1963) z57-I59
PN 10061
Induced, soluble phenylalanine hydroxylase from Pseudomonas sp. grown on phenylalanine or tyrosine T h e f o r m a t i o n of t y r o s i n e b y t h e p h e n y l a l a n i n e h y d r o x y l a s e (EC 1.99.1.2 ) of m a m m a l i a n liver has been well s t u d i e d z-4. T h e biosynthesis of t y r o s i n e in m i c r o o r g a n i s m s is g e n e r a l l y v i a an e n t i r e l y different p a t h w a y 5,6. A l t h o u g h it has been r e p o r t e d t h a t i n t a c t cells of certain species of m i c r o o r g a n i s m s can form t y r o s i n e from p h e n y l alanine u n d e r the p r o p e r conditions 7-9, t h e e n z y m e or enzymes i n v o l v e d h a v e n o t been o b t a i n e d in cell-free e x t r a c t s . This c o m m u n i c a t i o n describes a soluble, cell-free p r e p a r a t i o n from P s e u d o m o n a s sp. which h y d r o x y l a t e s p h e n y l a l a n i n e to form tyrosine. P s e u d o m o n a s sp. (ATCC I I 2 9 9 a ) was grown on m i n i m a l m e d i a c o n t a i n i n g 0 . 1 % K2HPO4, o . 1 % KH~PO4, 0.02% MgSO 4, 0 . 2 % NH4C1, a n d either 0 . 2 % t - p h e n y l a l a n i n e , 0 . 1 % L-tyrosine, or 0 . 2 % L-asparagine (pH 6.8). The cells were h a r v e s t e d d u r i n g log phase a n d washed five times w i t h 0 . 2 % p h o s p h a t e buffer c o n t a i n i n g 0.02% MgSO a (pH 6.8). The c o n c e n t r a t e d cell suspension was lysed in an osmotic pressure cell a n d the l y s a t e centrifuged in a Spinco centrifuge, Model L, at 4 ° 00o r e v . / m i n for I h. The clear s u p e r n a t a n t fraction from this p r e p a r a t i o n was used for these e x p e r i m e n t s . The s u p e r n a t a n t fraction was i n c u b a t e d in air a t 3 °0 w i t h D P N H a n d L-phenylalanine. A t the end of the i n c u b a t i o n period trichloroacetic acid was a d d e d to a final c o n c e n t r a t i o n of 60,/0. T y r o s i n O ° a n d p h e n y l a l a n i n e n were d e t e r m i n e d fluorom e t r i c a l l y in aliquots of t h e protein-free filtrate. H i g h - s p e e d s u p e r n a t a n t p r e p a r a t i o n s from cells grown on p h e n y l a l a n i n e conBiochim. Biophys. Acta, 77 (I963) I59 i6i
16o
PRELIMINARY NOTES
vetted phenylalanine to tyrosine (Table I). The reaction was completely dependent on exogenous phenylalanine and was stimulated by added DPNH. D-Phenylalanine was not hydroxylated. T P N H was inactive and, in fact, inhibited the reaction. The enzyme was saturated with respect to D P N H and phenylalanine at the concentrations used. The preparation did not metabolize tyrosine under these conditions. The conTABLE I TYROSINE
FORMATION BY FROM
HIGH-SPEED SUPERNATANT FRACTION PSEUDOMONAS SP.
T h e c o m p l e t e s y s t e m c o n s i s t e d of 2. 5 ~ m o l e s of D P N H , 6.06 ~ m o l e s of L - p h e n y l a l a n i n e , a n d 0. 5 m l of s u p e r n a t a n t in 0 . 2 % p h o s p h a t e buffer c o n t a i n i n g o 0 2 % MgSO~ (pH 6.8). The t o t a l v o l u m e w a s 0.8 ml. E x p e r i m e n t s I a n d 2 c o n t a i n e d 6.85 a n d 5.36 m g of p r o t e i n , r e s p e c t i v e l y . T h e r e a c t i o n m i x t u r e s were s h a k e n for 20 rain a t 30 ° a n d t h e r e a c t i o n s s t o p p e d w i t h 0.2 ml of 3O~'o t r i c h l o r o a c e t i c acid. p~ol~s Tyrosine Incubation
Complete -- D P N H -- P h e n y l a l a n i n e Enzyme C o m p l e t e ( e n z y m e b o i l e d 3 ° sec) C o m p l e t e (o min)
No. •
No. 2
1.8i 0.99 0.55 0.03 0.55 o.6t
t.23 o.8i 0.45 0.02 0.44 o.44
version of phenylalanine to tyrosine was stoichiometric and linear for at least 30 min. The formation of tyrosine was linear with enzyme concentration above a minimal protein content of about 2 mg per o.8-ml incubation. All the experiments presented were carried out with protein concentrations between 5 and 7 mg per incubation. The addition of components from the mammalian phenylalanine hydroxylase system, i.e., concentrates containing the pteridine cofactor or preparations of the "sheep enzyme 'q* had no effect on this activity. The relationship of this hydroxylase to the mammalian phenylalanine hydroxylase or to the other hydroxylases elaborated b y this organism13,14 remains to be studied. Extracts from cells grown on asparagine were devoid of hydroxylase activity (Table II). Extracts from cells grown on tyrosine, however, possessed about the same TABLE II TYROSINE
FORMATION BY HIGH-SPEED ON
SUPERNATANT
FRACTIONS FROM CELLS GROWN
VARIOUS MEDIA
T h e c o n d i t i o n s o f i n c u b a t i o n w e r e as i n d i c a t e d i n T a b l e I. F i g u r e s for t y r o s i n e f o r m a t i o n w e re o b t a i n e d b y s u b t r a c t i n g t h e a m o u n t s of t y r o s i n e in t h e z e r o - t i m e s a m p l e s f r o m t h a t i n t h e c omplete system. The n u m b e r s represent two separate e x p e r i m e n t s with each culture m e d i u m . Culture medium
L - P h e n y l a l a n i n e (0.2%) L-Tyrosine (o.i %) L - A s p a r a g i n e (0.2 %)
!~moles tyrosine formedlrag protein
O. 18, O.15 0.25, o.18 0.003, 0.003
Biochira.
Biophys.
Acta,
77 (1963) 159-161
PRELIMINARY NOTES
161
a m o u n t o f h y d r o x y l a s e a c t i v i t y as d i d e x t r a c t s o f cells g r o w n o n p h e n y l a l a n i n e . T h i s o b s e r v a t i o n h a s b e e n c o n f i r m e d in s t u d i e s o n t h e m e t a b o l i s m a n d i n c o r p o r a t i o n o f p h e n y l a l a n i n e in w h o l e cells 15. O t h e r s i m i l a r e x a m p l e s o f t h e i n d u c t i o n o f t h e i n i t i a l e n z y m e o f a d e g r a d a t i v e p a t h w a y b y t h e p r o d u c t o f its a c t i o n h a v e b e e n reported16,17. D a t a are a v a i l a b l e t o i n d i c a t e t h a t t h e t y r o s i n e f o r m e d b y t h i s i n d u c i b l e s y s t e m c a n b e u s e d for p r o t e i n s y n t h e s i s 15. A s t u d y o f t h e r e l a t i o n s h i p b e t w e e n t h e a l t e r n a t e r o u t e s o f t y r o s i n e f o r m a t i o n in t h e i n d u c e d o r g a n i s m is in progress.
LaOo,ato,y of Clinical Biochemistry, National Heart Institute, National Institutes of Health, Bethesda, Md. ( U . S . A . )
GORDON GUROFF TAKEO ITO*
1 s. UDENFRIEND AND J. R. COOPER, J. Biol. Chem., 194 (1952) 503 . 2 C. MITOMA, Arch. Biochem. Biophys., 6o (1956) 476. s S. KAffFMAN, J. Biol. Chem., 234 (1959) 26774 S. KAUFMAN,in O. 1-IAYAISHI,Oxygenases, Academic Press, New York, 1962, p. 129. s B. D. DAvis, J. Biol. Chem., 191 (I95I) 315 . 6 B. D. DAVIS, in W. D. MCELROY ANn H. B. GL&SS, Amino Acid Metabolism, Johns Hopkins Press, Baltimore, 1955, p. 7997 S. DAGLEY, M. E. FEWSTER, AND F. C. HAPPOLD, J. Gen. Microbiol., 8 (1953) I. s C. MITOMAAND L. C. LEEPER, Federa2ion Proc., 13 (1954) 266. 9 N. KUI~ITA, Med. J. Osaka Univ., 7 (1956) 2o3. 10 T. P. WAALKES AND S. UDENFRIEI~D. J. Lab. Clin. Med., 5° (1957) 733lz M. W. MCCAMAN AND E. ROBINS, J. Lab. Clin. Med., 59 (1962) 885. 12 S. KAUFMAN,in S. 19. COLOWICKANn N. O. KAPLAN, Methods in Enzymo!ogy, Vol. 5, Academic Press, New York, 1962, p. 800. lS S. ROTHBERG AND O. HAYAISHI, J. Biol. Chem., 229 (I957) 897. 14 H. TANIUCHI AND O. HAYAISHI, J. Biol. Chem., 238 (1963) 283. x5 T. ITO, G. GUROFF, AND S. UDENFRIEND, in preparation. l i t R. Y. STANIER ANn M. TSUCHIDA, J. BacterioL, 58 (1949) 451 7 M. SUDA, I. MIYAHARA, K. TOMIHATAAND A. KATO, Med. J. Osaka Univ., 3 (I952) 115R e c e i v e d M a y 27th, I 9 6 3
* Visiting Scientist from the Department of Biochemistry, Nihon University, School of Medicine, Tokyo, Japan.
Biochim. Biophys. Acta, 77 (I963) 159-16I
PN IOO62
The involvement of sulfhydryl sites in dopamine~-hydroxylase activity The enzyme dopamine-fl-hydroxylase catalyzes the fl-hydroxylation of dopamine t a n d o t h e r s t r u c t u r a l r e l a t e d p h e n y l e t h y l a m i n e s a n d p h e n y l p r o p y l a m i n e s 2-4. A s cofactors, ascorbic and fumaric acids are required. While it was shown that the e n z y m a t i c f l - h y d r o x y l a t i o n o f t h e a m i n e s is c o u p l e d t o a s t o i c h i o m e t r i c a l l y e q u i v a l e n t o x i d a t i o n o f a s c o r b i c acid, t h e r e q u i r e m e n t for f u m a r a t e is still u n e x p l a i n e d 1. T h e e n z y m a t i c h y d r o x y l a t i o n is s t i m u l a t e d b y A T P , a n d i t w a s s u g g e s t e d t h a t A T P f u n c t i o n s as a c h e l a t i n g a g e n t r a t h e r t h a n as a d o n o r o f h i g h e n e r g y z. E D T A in-
Biochim, Biophys, Aaa, 77 (1963) 161-164
PRELIMINARY NOTES
to be closely r e l a t e d t o - - i f n o t i d e n t i c a l w i t h - - t h e s y s t e m effecting aerobic h y d r o x y l a tion of a l k a n e s to give t h e p r i m a r y alcohol. Koninklijke/Shell Laboratorium, Amsterdam, Shell Internationale Research Maatschappij, N . V . , 's Gravenhage ( T h e Netherlands)
A. C. VAN DER LINDEN
1 G. J. E. THIJSSE AND A. C. VAN DER LINDEN, Antonie van Leeuwenhoek, 29 (z963) 89. E. R. LEADBETTER AND J. W. FOSTER, Arch. Microbiol., 35 (196o) 92. 3 J. E. STEWART, R. E. KALLIO, D. P. STEVENSON, A. C. JONES AND D. O. SCHISSLER, J.Bacteriol., 78 (I959) 441. J. B. DAVIS AND H. F. YARBROUGH, Science, 137 (1962) 615. J. CHOUTEAU, E. AZOULAYAND J. C. SENEZ, Nature, 194 (1962) 576. R. E. PARKER AND INT.S. ISAACS,Chem. Revs., (1959) 7377 B. i . BLOOM AND G. M. SHULL, J. Am. Chem. Soc.. 77 (1955) 5767 . s C. J. SIH, J. Bacteriol., 84 (1962) 382. 9 H. ICHIHARA, t(. ADACHI, K. HOSOKAWAAND Y. TAKEDA, J. Biol. Chem., 237 (1962) 2296. x0 H. S. MASON, Advan. Enzymol., 19 (I957) 79. R e c e i v e d A p r i l 24th, 1963 Biochim. Biophys. Acta, 77 (1963) z57-I59
PN 10061
Induced, soluble phenylalanine hydroxylase from Pseudomonas sp. grown on phenylalanine or tyrosine T h e f o r m a t i o n of t y r o s i n e b y t h e p h e n y l a l a n i n e h y d r o x y l a s e (EC 1.99.1.2 ) of m a m m a l i a n liver has been well s t u d i e d z-4. T h e biosynthesis of t y r o s i n e in m i c r o o r g a n i s m s is g e n e r a l l y v i a an e n t i r e l y different p a t h w a y 5,6. A l t h o u g h it has been r e p o r t e d t h a t i n t a c t cells of certain species of m i c r o o r g a n i s m s can form t y r o s i n e from p h e n y l alanine u n d e r the p r o p e r conditions 7-9, t h e e n z y m e or enzymes i n v o l v e d h a v e n o t been o b t a i n e d in cell-free e x t r a c t s . This c o m m u n i c a t i o n describes a soluble, cell-free p r e p a r a t i o n from P s e u d o m o n a s sp. which h y d r o x y l a t e s p h e n y l a l a n i n e to form tyrosine. P s e u d o m o n a s sp. (ATCC I I 2 9 9 a ) was grown on m i n i m a l m e d i a c o n t a i n i n g 0 . 1 % K2HPO4, o . 1 % KH~PO4, 0.02% MgSO 4, 0 . 2 % NH4C1, a n d either 0 . 2 % t - p h e n y l a l a n i n e , 0 . 1 % L-tyrosine, or 0 . 2 % L-asparagine (pH 6.8). The cells were h a r v e s t e d d u r i n g log phase a n d washed five times w i t h 0 . 2 % p h o s p h a t e buffer c o n t a i n i n g 0.02% MgSO a (pH 6.8). The c o n c e n t r a t e d cell suspension was lysed in an osmotic pressure cell a n d the l y s a t e centrifuged in a Spinco centrifuge, Model L, at 4 ° 00o r e v . / m i n for I h. The clear s u p e r n a t a n t fraction from this p r e p a r a t i o n was used for these e x p e r i m e n t s . The s u p e r n a t a n t fraction was i n c u b a t e d in air a t 3 °0 w i t h D P N H a n d L-phenylalanine. A t the end of the i n c u b a t i o n period trichloroacetic acid was a d d e d to a final c o n c e n t r a t i o n of 60,/0. T y r o s i n O ° a n d p h e n y l a l a n i n e n were d e t e r m i n e d fluorom e t r i c a l l y in aliquots of t h e protein-free filtrate. H i g h - s p e e d s u p e r n a t a n t p r e p a r a t i o n s from cells grown on p h e n y l a l a n i n e conBiochim. Biophys. Acta, 77 (I963) I59 i6i
16o
PRELIMINARY NOTES
vetted phenylalanine to tyrosine (Table I). The reaction was completely dependent on exogenous phenylalanine and was stimulated by added DPNH. D-Phenylalanine was not hydroxylated. T P N H was inactive and, in fact, inhibited the reaction. The enzyme was saturated with respect to D P N H and phenylalanine at the concentrations used. The preparation did not metabolize tyrosine under these conditions. The conTABLE I TYROSINE
FORMATION BY FROM
HIGH-SPEED SUPERNATANT FRACTION PSEUDOMONAS SP.
T h e c o m p l e t e s y s t e m c o n s i s t e d of 2. 5 ~ m o l e s of D P N H , 6.06 ~ m o l e s of L - p h e n y l a l a n i n e , a n d 0. 5 m l of s u p e r n a t a n t in 0 . 2 % p h o s p h a t e buffer c o n t a i n i n g o 0 2 % MgSO~ (pH 6.8). The t o t a l v o l u m e w a s 0.8 ml. E x p e r i m e n t s I a n d 2 c o n t a i n e d 6.85 a n d 5.36 m g of p r o t e i n , r e s p e c t i v e l y . T h e r e a c t i o n m i x t u r e s were s h a k e n for 20 rain a t 30 ° a n d t h e r e a c t i o n s s t o p p e d w i t h 0.2 ml of 3O~'o t r i c h l o r o a c e t i c acid. p~ol~s Tyrosine Incubation
Complete -- D P N H -- P h e n y l a l a n i n e Enzyme C o m p l e t e ( e n z y m e b o i l e d 3 ° sec) C o m p l e t e (o min)
No. •
No. 2
1.8i 0.99 0.55 0.03 0.55 o.6t
t.23 o.8i 0.45 0.02 0.44 o.44
version of phenylalanine to tyrosine was stoichiometric and linear for at least 30 min. The formation of tyrosine was linear with enzyme concentration above a minimal protein content of about 2 mg per o.8-ml incubation. All the experiments presented were carried out with protein concentrations between 5 and 7 mg per incubation. The addition of components from the mammalian phenylalanine hydroxylase system, i.e., concentrates containing the pteridine cofactor or preparations of the "sheep enzyme 'q* had no effect on this activity. The relationship of this hydroxylase to the mammalian phenylalanine hydroxylase or to the other hydroxylases elaborated b y this organism13,14 remains to be studied. Extracts from cells grown on asparagine were devoid of hydroxylase activity (Table II). Extracts from cells grown on tyrosine, however, possessed about the same TABLE II TYROSINE
FORMATION BY HIGH-SPEED ON
SUPERNATANT
FRACTIONS FROM CELLS GROWN
VARIOUS MEDIA
T h e c o n d i t i o n s o f i n c u b a t i o n w e r e as i n d i c a t e d i n T a b l e I. F i g u r e s for t y r o s i n e f o r m a t i o n w e re o b t a i n e d b y s u b t r a c t i n g t h e a m o u n t s of t y r o s i n e in t h e z e r o - t i m e s a m p l e s f r o m t h a t i n t h e c omplete system. The n u m b e r s represent two separate e x p e r i m e n t s with each culture m e d i u m . Culture medium
L - P h e n y l a l a n i n e (0.2%) L-Tyrosine (o.i %) L - A s p a r a g i n e (0.2 %)
!~moles tyrosine formedlrag protein
O. 18, O.15 0.25, o.18 0.003, 0.003
Biochira.
Biophys.
Acta,
77 (1963) 159-161
PRELIMINARY NOTES
161
a m o u n t o f h y d r o x y l a s e a c t i v i t y as d i d e x t r a c t s o f cells g r o w n o n p h e n y l a l a n i n e . T h i s o b s e r v a t i o n h a s b e e n c o n f i r m e d in s t u d i e s o n t h e m e t a b o l i s m a n d i n c o r p o r a t i o n o f p h e n y l a l a n i n e in w h o l e cells 15. O t h e r s i m i l a r e x a m p l e s o f t h e i n d u c t i o n o f t h e i n i t i a l e n z y m e o f a d e g r a d a t i v e p a t h w a y b y t h e p r o d u c t o f its a c t i o n h a v e b e e n reported16,17. D a t a are a v a i l a b l e t o i n d i c a t e t h a t t h e t y r o s i n e f o r m e d b y t h i s i n d u c i b l e s y s t e m c a n b e u s e d for p r o t e i n s y n t h e s i s 15. A s t u d y o f t h e r e l a t i o n s h i p b e t w e e n t h e a l t e r n a t e r o u t e s o f t y r o s i n e f o r m a t i o n in t h e i n d u c e d o r g a n i s m is in progress.
LaOo,ato,y of Clinical Biochemistry, National Heart Institute, National Institutes of Health, Bethesda, Md. ( U . S . A . )
GORDON GUROFF TAKEO ITO*
1 s. UDENFRIEND AND J. R. COOPER, J. Biol. Chem., 194 (1952) 503 . 2 C. MITOMA, Arch. Biochem. Biophys., 6o (1956) 476. s S. KAffFMAN, J. Biol. Chem., 234 (1959) 26774 S. KAUFMAN,in O. 1-IAYAISHI,Oxygenases, Academic Press, New York, 1962, p. 129. s B. D. DAvis, J. Biol. Chem., 191 (I95I) 315 . 6 B. D. DAVIS, in W. D. MCELROY ANn H. B. GL&SS, Amino Acid Metabolism, Johns Hopkins Press, Baltimore, 1955, p. 7997 S. DAGLEY, M. E. FEWSTER, AND F. C. HAPPOLD, J. Gen. Microbiol., 8 (1953) I. s C. MITOMAAND L. C. LEEPER, Federa2ion Proc., 13 (1954) 266. 9 N. KUI~ITA, Med. J. Osaka Univ., 7 (1956) 2o3. 10 T. P. WAALKES AND S. UDENFRIEI~D. J. Lab. Clin. Med., 5° (1957) 733lz M. W. MCCAMAN AND E. ROBINS, J. Lab. Clin. Med., 59 (1962) 885. 12 S. KAUFMAN,in S. 19. COLOWICKANn N. O. KAPLAN, Methods in Enzymo!ogy, Vol. 5, Academic Press, New York, 1962, p. 800. lS S. ROTHBERG AND O. HAYAISHI, J. Biol. Chem., 229 (I957) 897. 14 H. TANIUCHI AND O. HAYAISHI, J. Biol. Chem., 238 (1963) 283. x5 T. ITO, G. GUROFF, AND S. UDENFRIEND, in preparation. l i t R. Y. STANIER ANn M. TSUCHIDA, J. BacterioL, 58 (1949) 451 7 M. SUDA, I. MIYAHARA, K. TOMIHATAAND A. KATO, Med. J. Osaka Univ., 3 (I952) 115R e c e i v e d M a y 27th, I 9 6 3
* Visiting Scientist from the Department of Biochemistry, Nihon University, School of Medicine, Tokyo, Japan.
Biochim. Biophys. Acta, 77 (I963) 159-16I
PN IOO62
The involvement of sulfhydryl sites in dopamine~-hydroxylase activity The enzyme dopamine-fl-hydroxylase catalyzes the fl-hydroxylation of dopamine t a n d o t h e r s t r u c t u r a l r e l a t e d p h e n y l e t h y l a m i n e s a n d p h e n y l p r o p y l a m i n e s 2-4. A s cofactors, ascorbic and fumaric acids are required. While it was shown that the e n z y m a t i c f l - h y d r o x y l a t i o n o f t h e a m i n e s is c o u p l e d t o a s t o i c h i o m e t r i c a l l y e q u i v a l e n t o x i d a t i o n o f a s c o r b i c acid, t h e r e q u i r e m e n t for f u m a r a t e is still u n e x p l a i n e d 1. T h e e n z y m a t i c h y d r o x y l a t i o n is s t i m u l a t e d b y A T P , a n d i t w a s s u g g e s t e d t h a t A T P f u n c t i o n s as a c h e l a t i n g a g e n t r a t h e r t h a n as a d o n o r o f h i g h e n e r g y z. E D T A in-
Biochim, Biophys, Aaa, 77 (1963) 161-164