- Email: [email protected]
[5] Phenylalanine 4-monooxygenase from Chromobacterium violaceum
44
C A T A B O L I S M O F T H E A R O M A T I C A M I N O ACIDS
[5]
enzymes comigrate on dodecyl sulfate gel electrophoresis. No significant difference is observed between the amino acid composition of the bovine and the rat enzyme preparations or in the reactivity of their sulfhydryl groups, and both enzymes reveal the same concentration-dependent inhibition by bathophenanthroline disulfonate, but are not inhibited by bathocuproine disulfonate. Immunological studies have revealed cross-reactivity between the hydroxylases isolated from bovine and rat liver, indicating that the two enzymes have a similar antigenic structure as expected from the similarities in amino acid composition and other physicochemical properties. Both enzymes are phosphorylated by cyclic AMPdependent protein kinase. On the other hand, the specific activity of the bovine enzyme using 6,7-dimethyltetrahydropterin as the cofactor (90 to 240 mU/mg protein), 9 is markedly lower than that obtained for the rat enzyme (1300 to 1600 mU/mg protein). The basis of this difference in specific activities is not yet clear. Acknowledgments The workfromthe authors' laboratoryhas been supportedby the NorwegianCouncilfor Research on Mental Retardation, The Nordic Insulin Foundation,and the NorwegianResearch Councilfor Science and the Humanities.
[5] P h e n y l a l a n i n e 4 - M o n o o x y g e n a s e f r o m
Chromobacterium violaceum By HITOSHI FUJISAWA and HIROYASU NAKATA Phenylalanine 4-monooxygenase (hydroxylase) [L-phenylalanine, tetrahydropteridine : oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1] is a monooxygenase which catalyzes the conversion of Lphenylalanine to L-tyrosine using tetrahydrobiopterin as a reducing agent and molecular oxygen as an oxidizing agent. L-Phenylalanine+ tetrahydrobiopterin+ 02--> L-tyrosine+ dihydrobiopterin+ H20 Assay Method
Principle. The natural cofactor for phenylalanine hydroxylase is tetrahydrobiopterin I but the enzyme can use the commercially available corns.
K a n f m a n , Proc. Natl. Acad. ScL U.S.A. 50, I085 (1963).
METHODS IN ENZYMOLOGY, VOL. 142
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
[5]
PHENYLALANINE 4-MONOOXYGENASE FROM C. v i o l a c e u m
45
pounds, 2-amino-4-hydroxy-6-methyltetrahydropteridine and 2-amino-4hydroxy-6,7-dimethyltetrahydropteridine, as electron donors. In the assay used to follow the purification of the enzyme, 2-amino-4-hydroxy6,7-dimethyltetrahydropteridine is used as an electron donor and the tyrosine produced is determined fluorometrically according to the nitrosonaphthol procedure of Waalkes and Udenfriend 2 as modified by Woo et al. 3
Reagents Tris-HC1 buffer, 0.5 M, pH 7.3 at 24° Catalase, 20 mg/ml (Boehringer) Dithiothreitol, 50 mM L-Phenylalanine, 25 mM 2-Amino-4-hydroxy-6,7-dimethyltetrahydropteridine, l0 mM in l0 mM HCI Trichloroacetic acid, 30% Nitrosonaphthol solution: mix equal volumes of 1.5 M HNO3, 0.1 M NaNO2, and 1.3 mg/ml 1-nitroso-2-naphthol in absolute ethanol; this solution must be prepared immediately before use 1,2-Dichloroethane Procedure. The assay mixture contains 50/zl of 0.5 M Tris-HCl buffer (pH 7.3), 5/xl of 20 mg/ml catalase, 20/xl of 50 mM dithiothreitol, 20/xl of 50 mM phenylalanine, l0/~l of l0 mM 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine, and a suitable amount of the sample to be assayed, in a final volume of 250/xl. The reaction is started by the addition of 2-amino4-hydroxy-6,7-dimethyltetrahydropteridine, done at 24° for 5 min with mild shaking, and stopped by the addition of 50/zl of 30% trichloroacetic acid. Immediately thereafter, 0.5 ml of nitrosonaphthol solution is added and the mixture is placed in a water bath at 60° for 20 min. After cooling, 2.5 ml of water and 5 ml of dichloroethane are added, and the mixture is shaken vigorously. After centrifugation, the supernatant aqueous layer is transferred to a cuvette and the fluorescence is measured at an excitation wavelength of 435 nm and an emission wavelength of 540 nm with a spectrofluorometer. The absolute quantity of tyrosine formed can be determined by comparing the fluorescence measurements with a standard tyrosine curve. Definition o f Unit and Specific Activity. One unit of enzyme is defined as the amount that produces 1/xmol of tyrosine per min at 24°. Specific activity is defined as the number of enzyme units per mg of protein. 2 T. P. Waalkes and S. Undenfriend, J. Lab. Clin. Med. 50, 733 (1957). 3 S. L. C. Woo, S. S. Gillam, and L. I. Woolf, Biochem. J. 139~ 741 (1974).
46
CATABOLISM OF THE AROMATIC AMINO ACIDS
[5]
Protein is determined by the method of Lowry et al. 4 with bovine serum albumin as a standard. Purification 5 Chromobacterium violaceum is grown in a medium containing Lphenylalanine as an inducer at 37 ° with vigorous aeration. The medium contains, in a volume of I liter, L-phenylalanine, 5 g; meat extract, 5 g; peptone, 15 g; NaCI, 5 g; Na2HPO4, 3 g; and NaH2PO4, 1.5 g; the pH is adjusted to 7. The cells are harvested when growth reaches the stationary phase and are stored at - 2 0 ° with little loss of activity for a month. The yield of the cells is approximately 10 g wet weight per liter. When this organism is grown in a medium containing L-tryptophan or L-tyrosine in place of L-phenylalanine, no significant activity of phenylalanine hydroxylase is observed. When this organism is grown in a medium containing 0.5% L-phenylalanine as a sole carbon source, the yield of the cells is only 20% of that of the cells grown in the rich medium described above. All subsequent extract and purification procedures are carried out at 4 ° and centrifugation is carried out at 27,000 g for 20 min unless otherwise stated. Step 1. Crude Extract. Harvested cells (330 g) are suspended in 500 ml of 50 m M acetate buffer (pH 6.0) and are disrupted in 45-ml batches by sonic oscillation with a 9-kHz sonic oscillator for 15 min. The residue is removed by centrifugation. Step 2. Protamine Treatment. To the supernatant fluid (620 ml), 25 ml of 5% protamine sulfate solution is added with stirring. After stirring for 25 min, the precipitate is removed by centrifugation. The supernatant solution is dialyzed overnight against 10 liters of 50 mM acetate buffer (pH 6.0). The insoluble material is removed by centrifugation. Step 3. DEAE-Sephadex A-50 Treatment. To the supernatant solution (670 ml), 500 ml of hydrated DEAE-Sephadex A-50 equilibrated with 50 m M acetate buffer (pH 6.0) is added, and the mixture is stirred gently for 10 hr. The resin containing the active enzyme is washed with 1 liter of the equilibration buffer and then with 1 liter of 50 mM acetate buffer (pH 6.0) containing 0.1 M KCI on a Biichner funnel. The resin is then suspended in 700 ml of 50 m M acetate buffer (pH 6.0) containing 0.6 M KCI. After stirring for 30 min, the mixture is filtered on a Biichner funnel. The resin is again suspended in 350 ml of 50 m M acetate buffer (pH 6.0) containing 0.6 4 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 5 H. Nakata, T. Yamauchi, and H. Fujisawa, J. Biol. Chem. 254, 1829 (1979).
[5]
PHENYLALANINE 4-MONOOXYGENASE FROM C. violaceum
47
M KCI and the mixture is filtered as before. The combined filtrate is brought to 70% saturation with solid ammonium sulfate and stirred for 20 min. The mixture is centrifuged and the resulting precipitate is dissolved in a minimum volume of 50 mM acetate buffer (pH 6.0). Step 4. Acid Treatment. The pH of the enzyme solution (80 ml) from Step 3 is adjusted to 4.9 by the addition of I N acetic acid and the resulting precipitate is immediately removed by centrifugation at 27,000 g for 5 min. The supernatant solution is neutralized by the addition of 1 M Tris and then dialyzed against 2 liters of 50 mM acetate buffer (pH 6.0) overnight. Step 5. DEAE-Cellulose Chromatography. The dialyzate (110 ml) is applied to a DEAE-cellulose (DE-52) column (2 × 10 cm) which has been equilibrated with 50 m M acetate buffer (pH 6.0). After washing with about 200 ml of the equilibration buffer, the column is eluted with a 400 ml linear gradient from 0 to 0.3 M KCI in 50 mM acetate buffer (pH 6.0) at a flow rate of 25 ml/hr and fractions are collected every 10 ml. Active fractions which should be eluted from the column after 160 ml, in a total volume of about 80 ml, are pooled and precipitated by the addition of solid ammonium sulfate (70% saturation). The resulting precipitate is dissolved in 50 m M acetate buffer (pH 6.0). Step 6. First Ultrogel AcA 44 Gel Filtration. The active solution (10 ml) from Step 5 is layered onto an Ultrogel AcA 44 column (2 × 100 cm) equilibrated with 50 m M acetate buffer (pH 6.0) and is eluted with the equilibration buffer. Fractions of 5 ml are collected at a flow rate of 15 ml/ hr. The active fractions which usually emerge after 185 ml, in a total volume of 30 ml, are pooled and precipitated by the addition of solid ammonium sulfate (70% saturation). The resulting precipitate is dissolved in 50 m M acetate buffer (pH 6.0). Step 7. Second Ultrogel AcA 44 Gel Filtration. The concentrated enzyme solution (5 ml) from Step 6 is layered onto the same column that is used at Step 6. Fractions of 5 ml are collected at a flow rate of 10 ml/hr and the active fractions which usually emerge after 190 ml, in a total volume of 25 ml, are pooled. Step 8. Hydroxylapatite Chromatography. The enzyme solution from the previous step is adsorbed on a hydroxylapatite column (2 × 4 cm) which has been equilibrated with 50 m M acetate buffer (pH 6.0). After washing with 100 ml of the equilibration buffer, the column is eluted with 100 ml of 10 m M sodium phosphate buffer (pH 7.0) and fractions of 10 ml are collected at a flow rate of 20 ml/hr. Active fraction are pooled and precipitated by the addition of solid ammonium sulfate (70% saturation). The precipitate is dissolved in 50 mM acetate buffer (pH 6.0) and is dialyzed overnight, against the same buffer.
48
CATABOLISM OF THE AROMATIC AMINO ACIDS
[5]
TABLE I PURIFICATION OF PHENYLALANINE HYDROXYLASE FROM Chromobacteriurn violaceum
Purification step 1. 2. 3. 4. 5. 6. 7. 8. 9.
Sonic extract Protamine treatment DEAE-Sephadex A-50 Acid treatment DEAE-cellulose First Ultrogel AcA 44 Second Ultrogel AcA 44 Hydroxylapatite Blue dextran-phenylbutylamine-Sepharose
Protein (mg) 49,600 50,000 5,100 2,080 366 60 45 5.0 2.2
Specific Activity activity (units) (units/mg) 219 202 60.0 56.5 98.6 90.0 85.4 42.5 29.1
0.00442 0.00404 0.0118 0.0272 0.269 1.50 1.90 8.50 13.2
Yield (%)
Purification (fold)
100 92 27 26 45 41 39 19 13
1 1 3 6 61 339 430 1920 2990
Step 9. Blue Dextran-Phenylbutylamine-Sepharose Chromatography. The enzyme solution (20 ml) from Step 8 is loaded onto a Blue Dextranphenylbutylamine-Sepharose column (1.5 x 7.0 cm) equilibrated with 50 m M acetate buffer (pH 6.0). After washing with about 100 ml of the equilibration buffer, the column is eluted with a 200 ml linear gradient from 0 to 0.8 M KCI in 50 mM acetate buffer (pH 6.0) at a flow rate of 30 ml/hr and fractions are collected every 7 ml. Active fractions which usually emerge after 49 ml, inoa total volume of 84 ml, are pooled and precipitated by the addition of solid ammonium sulfate (70% saturation). The precipitate is dissolved in a minimum volume of 50 mM acetate buffer (pH 6.0). The purified enzyme can be stored at - 2 0 ° for 3 months with a loss of under 20% of the activity. The results of a typical purification are summarized in Table I. Properties 5
Purity. The purified enzyme gives a single protein band upon disc gel electrophoresis in the presence or absence of 0. I% sodium dodecyl sulfate. Physical Properties. The values of the molecular weight of the enzyme estimated by a number of techniques range from 31,200 to 32,400 and these are consistent with the value of 33,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme is a single polypeptide chain. The isoelectric point of the enzyme is determined to be 4.5 by polyacrylamide gel electrofocusing.
[5]
PHENYLALANINE 4-MONOOXYGENASE FROM C. violaceum
49
T A B L E II AMINO ACID COMPOSITION OF PHENYLALANINE HYDROXYLASE FROM Chromobacterium
violaceum
Amino acid
Mol%
Residues/ molecule"
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
3.6 1.9 5.5 13.6 4.5 3.0 7.6 6.8 6.6 10. I I. I 6.6 2. I 4. I 12.0 4.0 5.6 1.3
10.4 5.4 15.8 38.9 12.9 8.7 21.8 19.4 19.0 29.0 3.1 18.8 5.9 11.7 34.4 11.3 15.9 3.7
" Estimated by assuming a molecular weight of 32,000.
Chemical Properties. The amino acid composition of the enzyme is given in Table II. Analysis by the atomic absorption spectrophotometer shows that the enzyme contains no detectable amounts of iron. Catalytic Properties. The optimum pH for the enzyme activity is in the range of 7.3 to 7.5. The apparent Km values for L-phenylalanine and 2amino-4-hydroxy-6,7-dimethyltetrahydropteridine are approximately 140 and 54/xM, respectively. The apparent Vmax value is about 14/zmol of tyrosine/min/mg of protein. The rate of hydroxylation of L-tryptophan is about 0.4% of that of L-phenylalanine. With L-tyrosine, no detectable hydroxylation is observed. 2-Amino-4-hydroxy-6,7-dimethyltetrahydropteridine, 2-amino-4-hydroxy-6-methyltetrahydropteridine, and tetrahydrofolate serve as electron donors for the enzyme. Among these three synthetic pteridines, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine is the most active. The enzyme activity with 2-amino-4-hydroxy-6-methyltetrahydropteridine and tetrahydrofolate is approximately 60 and 10% of that with 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine, respectively.
C A T A B O L I S M O F T H E A R O M A T I C A M I N O ACIDS
[5]
enzymes comigrate on dodecyl sulfate gel electrophoresis. No significant difference is observed between the amino acid composition of the bovine and the rat enzyme preparations or in the reactivity of their sulfhydryl groups, and both enzymes reveal the same concentration-dependent inhibition by bathophenanthroline disulfonate, but are not inhibited by bathocuproine disulfonate. Immunological studies have revealed cross-reactivity between the hydroxylases isolated from bovine and rat liver, indicating that the two enzymes have a similar antigenic structure as expected from the similarities in amino acid composition and other physicochemical properties. Both enzymes are phosphorylated by cyclic AMPdependent protein kinase. On the other hand, the specific activity of the bovine enzyme using 6,7-dimethyltetrahydropterin as the cofactor (90 to 240 mU/mg protein), 9 is markedly lower than that obtained for the rat enzyme (1300 to 1600 mU/mg protein). The basis of this difference in specific activities is not yet clear. Acknowledgments The workfromthe authors' laboratoryhas been supportedby the NorwegianCouncilfor Research on Mental Retardation, The Nordic Insulin Foundation,and the NorwegianResearch Councilfor Science and the Humanities.
[5] P h e n y l a l a n i n e 4 - M o n o o x y g e n a s e f r o m
Chromobacterium violaceum By HITOSHI FUJISAWA and HIROYASU NAKATA Phenylalanine 4-monooxygenase (hydroxylase) [L-phenylalanine, tetrahydropteridine : oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1] is a monooxygenase which catalyzes the conversion of Lphenylalanine to L-tyrosine using tetrahydrobiopterin as a reducing agent and molecular oxygen as an oxidizing agent. L-Phenylalanine+ tetrahydrobiopterin+ 02--> L-tyrosine+ dihydrobiopterin+ H20 Assay Method
Principle. The natural cofactor for phenylalanine hydroxylase is tetrahydrobiopterin I but the enzyme can use the commercially available corns.
K a n f m a n , Proc. Natl. Acad. ScL U.S.A. 50, I085 (1963).
METHODS IN ENZYMOLOGY, VOL. 142
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
[5]
PHENYLALANINE 4-MONOOXYGENASE FROM C. v i o l a c e u m
45
pounds, 2-amino-4-hydroxy-6-methyltetrahydropteridine and 2-amino-4hydroxy-6,7-dimethyltetrahydropteridine, as electron donors. In the assay used to follow the purification of the enzyme, 2-amino-4-hydroxy6,7-dimethyltetrahydropteridine is used as an electron donor and the tyrosine produced is determined fluorometrically according to the nitrosonaphthol procedure of Waalkes and Udenfriend 2 as modified by Woo et al. 3
Reagents Tris-HC1 buffer, 0.5 M, pH 7.3 at 24° Catalase, 20 mg/ml (Boehringer) Dithiothreitol, 50 mM L-Phenylalanine, 25 mM 2-Amino-4-hydroxy-6,7-dimethyltetrahydropteridine, l0 mM in l0 mM HCI Trichloroacetic acid, 30% Nitrosonaphthol solution: mix equal volumes of 1.5 M HNO3, 0.1 M NaNO2, and 1.3 mg/ml 1-nitroso-2-naphthol in absolute ethanol; this solution must be prepared immediately before use 1,2-Dichloroethane Procedure. The assay mixture contains 50/zl of 0.5 M Tris-HCl buffer (pH 7.3), 5/xl of 20 mg/ml catalase, 20/xl of 50 mM dithiothreitol, 20/xl of 50 mM phenylalanine, l0/~l of l0 mM 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine, and a suitable amount of the sample to be assayed, in a final volume of 250/xl. The reaction is started by the addition of 2-amino4-hydroxy-6,7-dimethyltetrahydropteridine, done at 24° for 5 min with mild shaking, and stopped by the addition of 50/zl of 30% trichloroacetic acid. Immediately thereafter, 0.5 ml of nitrosonaphthol solution is added and the mixture is placed in a water bath at 60° for 20 min. After cooling, 2.5 ml of water and 5 ml of dichloroethane are added, and the mixture is shaken vigorously. After centrifugation, the supernatant aqueous layer is transferred to a cuvette and the fluorescence is measured at an excitation wavelength of 435 nm and an emission wavelength of 540 nm with a spectrofluorometer. The absolute quantity of tyrosine formed can be determined by comparing the fluorescence measurements with a standard tyrosine curve. Definition o f Unit and Specific Activity. One unit of enzyme is defined as the amount that produces 1/xmol of tyrosine per min at 24°. Specific activity is defined as the number of enzyme units per mg of protein. 2 T. P. Waalkes and S. Undenfriend, J. Lab. Clin. Med. 50, 733 (1957). 3 S. L. C. Woo, S. S. Gillam, and L. I. Woolf, Biochem. J. 139~ 741 (1974).
46
CATABOLISM OF THE AROMATIC AMINO ACIDS
[5]
Protein is determined by the method of Lowry et al. 4 with bovine serum albumin as a standard. Purification 5 Chromobacterium violaceum is grown in a medium containing Lphenylalanine as an inducer at 37 ° with vigorous aeration. The medium contains, in a volume of I liter, L-phenylalanine, 5 g; meat extract, 5 g; peptone, 15 g; NaCI, 5 g; Na2HPO4, 3 g; and NaH2PO4, 1.5 g; the pH is adjusted to 7. The cells are harvested when growth reaches the stationary phase and are stored at - 2 0 ° with little loss of activity for a month. The yield of the cells is approximately 10 g wet weight per liter. When this organism is grown in a medium containing L-tryptophan or L-tyrosine in place of L-phenylalanine, no significant activity of phenylalanine hydroxylase is observed. When this organism is grown in a medium containing 0.5% L-phenylalanine as a sole carbon source, the yield of the cells is only 20% of that of the cells grown in the rich medium described above. All subsequent extract and purification procedures are carried out at 4 ° and centrifugation is carried out at 27,000 g for 20 min unless otherwise stated. Step 1. Crude Extract. Harvested cells (330 g) are suspended in 500 ml of 50 m M acetate buffer (pH 6.0) and are disrupted in 45-ml batches by sonic oscillation with a 9-kHz sonic oscillator for 15 min. The residue is removed by centrifugation. Step 2. Protamine Treatment. To the supernatant fluid (620 ml), 25 ml of 5% protamine sulfate solution is added with stirring. After stirring for 25 min, the precipitate is removed by centrifugation. The supernatant solution is dialyzed overnight against 10 liters of 50 mM acetate buffer (pH 6.0). The insoluble material is removed by centrifugation. Step 3. DEAE-Sephadex A-50 Treatment. To the supernatant solution (670 ml), 500 ml of hydrated DEAE-Sephadex A-50 equilibrated with 50 m M acetate buffer (pH 6.0) is added, and the mixture is stirred gently for 10 hr. The resin containing the active enzyme is washed with 1 liter of the equilibration buffer and then with 1 liter of 50 mM acetate buffer (pH 6.0) containing 0.1 M KCI on a Biichner funnel. The resin is then suspended in 700 ml of 50 m M acetate buffer (pH 6.0) containing 0.6 M KCI. After stirring for 30 min, the mixture is filtered on a Biichner funnel. The resin is again suspended in 350 ml of 50 m M acetate buffer (pH 6.0) containing 0.6 4 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 5 H. Nakata, T. Yamauchi, and H. Fujisawa, J. Biol. Chem. 254, 1829 (1979).
[5]
PHENYLALANINE 4-MONOOXYGENASE FROM C. violaceum
47
M KCI and the mixture is filtered as before. The combined filtrate is brought to 70% saturation with solid ammonium sulfate and stirred for 20 min. The mixture is centrifuged and the resulting precipitate is dissolved in a minimum volume of 50 mM acetate buffer (pH 6.0). Step 4. Acid Treatment. The pH of the enzyme solution (80 ml) from Step 3 is adjusted to 4.9 by the addition of I N acetic acid and the resulting precipitate is immediately removed by centrifugation at 27,000 g for 5 min. The supernatant solution is neutralized by the addition of 1 M Tris and then dialyzed against 2 liters of 50 mM acetate buffer (pH 6.0) overnight. Step 5. DEAE-Cellulose Chromatography. The dialyzate (110 ml) is applied to a DEAE-cellulose (DE-52) column (2 × 10 cm) which has been equilibrated with 50 m M acetate buffer (pH 6.0). After washing with about 200 ml of the equilibration buffer, the column is eluted with a 400 ml linear gradient from 0 to 0.3 M KCI in 50 mM acetate buffer (pH 6.0) at a flow rate of 25 ml/hr and fractions are collected every 10 ml. Active fractions which should be eluted from the column after 160 ml, in a total volume of about 80 ml, are pooled and precipitated by the addition of solid ammonium sulfate (70% saturation). The resulting precipitate is dissolved in 50 m M acetate buffer (pH 6.0). Step 6. First Ultrogel AcA 44 Gel Filtration. The active solution (10 ml) from Step 5 is layered onto an Ultrogel AcA 44 column (2 × 100 cm) equilibrated with 50 m M acetate buffer (pH 6.0) and is eluted with the equilibration buffer. Fractions of 5 ml are collected at a flow rate of 15 ml/ hr. The active fractions which usually emerge after 185 ml, in a total volume of 30 ml, are pooled and precipitated by the addition of solid ammonium sulfate (70% saturation). The resulting precipitate is dissolved in 50 m M acetate buffer (pH 6.0). Step 7. Second Ultrogel AcA 44 Gel Filtration. The concentrated enzyme solution (5 ml) from Step 6 is layered onto the same column that is used at Step 6. Fractions of 5 ml are collected at a flow rate of 10 ml/hr and the active fractions which usually emerge after 190 ml, in a total volume of 25 ml, are pooled. Step 8. Hydroxylapatite Chromatography. The enzyme solution from the previous step is adsorbed on a hydroxylapatite column (2 × 4 cm) which has been equilibrated with 50 m M acetate buffer (pH 6.0). After washing with 100 ml of the equilibration buffer, the column is eluted with 100 ml of 10 m M sodium phosphate buffer (pH 7.0) and fractions of 10 ml are collected at a flow rate of 20 ml/hr. Active fraction are pooled and precipitated by the addition of solid ammonium sulfate (70% saturation). The precipitate is dissolved in 50 mM acetate buffer (pH 6.0) and is dialyzed overnight, against the same buffer.
48
CATABOLISM OF THE AROMATIC AMINO ACIDS
[5]
TABLE I PURIFICATION OF PHENYLALANINE HYDROXYLASE FROM Chromobacteriurn violaceum
Purification step 1. 2. 3. 4. 5. 6. 7. 8. 9.
Sonic extract Protamine treatment DEAE-Sephadex A-50 Acid treatment DEAE-cellulose First Ultrogel AcA 44 Second Ultrogel AcA 44 Hydroxylapatite Blue dextran-phenylbutylamine-Sepharose
Protein (mg) 49,600 50,000 5,100 2,080 366 60 45 5.0 2.2
Specific Activity activity (units) (units/mg) 219 202 60.0 56.5 98.6 90.0 85.4 42.5 29.1
0.00442 0.00404 0.0118 0.0272 0.269 1.50 1.90 8.50 13.2
Yield (%)
Purification (fold)
100 92 27 26 45 41 39 19 13
1 1 3 6 61 339 430 1920 2990
Step 9. Blue Dextran-Phenylbutylamine-Sepharose Chromatography. The enzyme solution (20 ml) from Step 8 is loaded onto a Blue Dextranphenylbutylamine-Sepharose column (1.5 x 7.0 cm) equilibrated with 50 m M acetate buffer (pH 6.0). After washing with about 100 ml of the equilibration buffer, the column is eluted with a 200 ml linear gradient from 0 to 0.8 M KCI in 50 mM acetate buffer (pH 6.0) at a flow rate of 30 ml/hr and fractions are collected every 7 ml. Active fractions which usually emerge after 49 ml, inoa total volume of 84 ml, are pooled and precipitated by the addition of solid ammonium sulfate (70% saturation). The precipitate is dissolved in a minimum volume of 50 mM acetate buffer (pH 6.0). The purified enzyme can be stored at - 2 0 ° for 3 months with a loss of under 20% of the activity. The results of a typical purification are summarized in Table I. Properties 5
Purity. The purified enzyme gives a single protein band upon disc gel electrophoresis in the presence or absence of 0. I% sodium dodecyl sulfate. Physical Properties. The values of the molecular weight of the enzyme estimated by a number of techniques range from 31,200 to 32,400 and these are consistent with the value of 33,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme is a single polypeptide chain. The isoelectric point of the enzyme is determined to be 4.5 by polyacrylamide gel electrofocusing.
[5]
PHENYLALANINE 4-MONOOXYGENASE FROM C. violaceum
49
T A B L E II AMINO ACID COMPOSITION OF PHENYLALANINE HYDROXYLASE FROM Chromobacterium
violaceum
Amino acid
Mol%
Residues/ molecule"
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
3.6 1.9 5.5 13.6 4.5 3.0 7.6 6.8 6.6 10. I I. I 6.6 2. I 4. I 12.0 4.0 5.6 1.3
10.4 5.4 15.8 38.9 12.9 8.7 21.8 19.4 19.0 29.0 3.1 18.8 5.9 11.7 34.4 11.3 15.9 3.7
" Estimated by assuming a molecular weight of 32,000.
Chemical Properties. The amino acid composition of the enzyme is given in Table II. Analysis by the atomic absorption spectrophotometer shows that the enzyme contains no detectable amounts of iron. Catalytic Properties. The optimum pH for the enzyme activity is in the range of 7.3 to 7.5. The apparent Km values for L-phenylalanine and 2amino-4-hydroxy-6,7-dimethyltetrahydropteridine are approximately 140 and 54/xM, respectively. The apparent Vmax value is about 14/zmol of tyrosine/min/mg of protein. The rate of hydroxylation of L-tryptophan is about 0.4% of that of L-phenylalanine. With L-tyrosine, no detectable hydroxylation is observed. 2-Amino-4-hydroxy-6,7-dimethyltetrahydropteridine, 2-amino-4-hydroxy-6-methyltetrahydropteridine, and tetrahydrofolate serve as electron donors for the enzyme. Among these three synthetic pteridines, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine is the most active. The enzyme activity with 2-amino-4-hydroxy-6-methyltetrahydropteridine and tetrahydrofolate is approximately 60 and 10% of that with 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine, respectively.