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[84] Iodination of tyrosine by chloroperoxidase; preparation of chloroperoxidase (Caldariomyces fumago)
648
AROMATIC AMINO ACIDS
[84]
pH Optimum. The pH optimum for the enzyme in 0.05 M Tris buffer is 8.2.
Specificity. The enzyme oxygenates 3,4-dihydroxyphenylacetic acid (1), ~ 3,4-dihydroxyphenylpropionic acid (1/35), 7 3,4-dihydroxybenzoic acid (1/1,200), * and catechol (1/18,000), 7 but does not oxygenate dopamine. Activators and Inhibitors. The enzyme is a simple protein which contains 5 atoms of iron as the sole prosthetic group and 16 residues of sulfhydryl groups per mole of the enzyme. Fe 2+ serves as an activator to enzyme preparations inactivated either by prolonged storage under air or treatment with hydrogen peroxide, and as a stabilizer to the native enzyme. The participation of the enzyme bound iron in the reaction, and its valency changes from ferrous to ferric are demonstrated by the ESR studies of the reaction, s The enzyme is inhibited competitively by 0-phenanthroline and 8-hydroxyquinoline, and noncompetitively by Hg 2+ and PCMB. The treatment of the enzyme with 8 equivalents of PCMB causes the dissociation of the enzyme into 4 subunits, which are inactive. 7Numbers in parentheses represent relative rates of oxidation. SH. Kita, M. Kamimoto, and S. Senoh, 7th Intern. Congr. Biochem., Tokyo, Abstr. IV-F-87 (1967).
[84] Iodination of Tyrosine by Chloroperoxidase; Preparation of Chloroperoxidase (Caldariomyces fumago) By LOWELL P. HAGER x - + H202 + H-acceptor --* X-acceptor + O H - + HzO
(1)
Chloroperoxidase catalyzes peroxidative halogenation reactions, with chloride, bromide, and iodide ions serving as the halogen anion donor. TM The reaction is relatively nonspecific with respect to the acceptor molecule. Any good nucleophile appears to possess acceptor activity; /3-keto acids and/3-diketones, by virtue of having an enolizable proton, are extremely active acceptors. Halide ions, especially iodide ion, are also good acceptors. Thus, under appropriate conditions, chloroperoxi1See articles [85a] and [85b] for a description of the iodination of tyrosine catalyzed by bovine lactoperoxidase and pig thyroid peroxidase. laL. P. Hager, D. R. Morris, F. S. Brown, and H. Eberwein,J. Biol. Chem. 241, 1769 (1966).
[84]
CHLOROPEROXIDASE
649
dase will catalyze the peroxidative formation of molecular iodine, bromine, and chlorine according to Eq. (2). 2x-~- H202 ---, X2 + 2OH-
(2)
Aromatic compounds possessing enolic character also serve as acceptors 2 although the overall rate of the halogenation reaction is variable and severalfold lower, with tyrosine, anisole, and benzyl alcohol serving as acceptors in decreasing order of reactivity. In the special case when iodide serves as the halogen donor and tyrosine serves as the halogen acceptor, it can be readily shown that molecular iodine is an obligate intermediate in the reaction. This follows from the fact that iodide ion is a much better acceptor in Eq. (1) than is tyrosine. Thus, the initial peroxidation of iodide ion produces elemental iodine. The subsequent utilization of molecular iodine leads to tyrosine iodination according to Eq. (3). Iz + H202 + tyrosine ~
2 monoiodotyrosine + H20
(3)
Crystalline chloroperoxidase preparations catalyze both reactions illustrated in Eqs. 2 and 3. Chloroperoxidase, a glycoprotein, is excreted in large amounts by Caldariomycesfumago in its phase of secondary metabolism. After an initial growth period in shake flasks of 6-7 days, chloroperoxidase excretion commences and continues for a 3-4-day period. Chloroperoxidase concentrations in the culture medium reach levels as high as 100 mg per liter of culture medium. Assay Method
The tyrosine iodination assay method for chloroperoxidase is based on the loss of tyrosine fluorescence in the conversion of tyrosine to its mol,oiodo derivative. The complete reaction mixture contains 300 micromoles of potassium phosphate buffer, pH 2.75, za 1 micromole o f tyrosine, 1.5 micromoles of hydrogen peroxide, 1 micromole of potassium iodide, and enzyme in total volume of 3 ml. The reaction is initiated by the addition of enzyme. Tyrosine fluorescence is measured at 303 m/.~ after irradiation at 275 m/~. There is an initial rapid decrease in fluorescence yield which is due to the formation of molecular iodine, which acts as screen and decreases the 275 m/~ irradiation. Subsequent utilization of the molecular iodine for the iodination of tyrosine yields 2F. S. Brown and L. P. Hager, MeetingAm. Chem. Soc., New York (1966). 2aThe pH 2.75 buffer is prepared by mixing 0.1 M HaPO4 and 0.1 M KH2PO4. The nonenzymatic iodination of tyrosine by 12 at pH 2.75 is so slow that it is of no quantitative significance.
650
AROMATIC AMINO ACIDS
[84]
a decrease in fluorescence that is proportional to the enzyme concentration. Routine assay of chloroperoxidase can be more easily accomplished in a reaction that measures the rate of halogenation of monochlorodimedone. The monochlorodimedone assay3 for chloroperoxidase is based on the loss of absorbance at 278 m/z that accompanies the conversion of 1,1-dimethyl-4-chloro-3,5-cyclohexanedione (monochlorodimedone) to 1,1-dimethyl-4,4-dichloro-3,5-cyclohexanedione (dichlorodimedone). The assay mixture contains 300 micromoles of potassium phosphate buffer, pH 2.75, 60 micromoles of potassium chloride, 6 micromoles of hydrogen peroxide, 0.3 micromole of monochlorodimedone, and a suitable aliquot of enzyme in a total volume of 3 ml. The reaction is started by the addition of enzyme. The change in absorbance at 278 m/J, as a function of time is recorded. The rate of the reaction is linear for 3-4 minutes. One unit of chloroperoxidase activity has been defined as the formation of 1 micromole of dichlorodimedone per minute under these assay conditions. Since the molar extinction coefficient of monochlorodimedone at 278 m/~ is 1.22 x 104, and dichlorodimedone has negligible absorption at this wavelength, the absorbancy change per minute times 0.246 is equal to 1 enzyme unit. Specific activity is defined as units per milligram of protein.
Preparation of the Enzyme Growth Medium. T h e mold is grown in a medium containing 4% glucose, 2% malt extract, 0.2% NaNO3, 0.2% KCI, 0.2% KH2PO4; 0.1% MgSO4" 7HzO, and 0.002% FeSO4.7H~O. One-liter Erlenmeyer flasks containing 600 ml of growth medium are incubated at 23-24 ° on a rotary shaker (New Brunswick, Model V) at 240 rpm. Chloroperoxidase excretion commences after approximately 7 days' incubation. Enzyme production usually reaches a maximum after 10-12 days' incubation. Chloroperoxidase excretion usually falls between the limits of 20 and 100 mg enzyme per liter of culture medium. lnoculum. Caldariomycesfumago (ATCC 16373) is maintained at liquid nitrogen temperature as a mycelial suspension frozen in 10% glycerolwater. The following procedure has been developed by Mrs. Frances A r n o w , Curator, Squibb Culture Collection, for stock culture maintenance. A slant of C. fumago is prepared on potato-dextrose agar and incubated 10-14 days at 25 °. With a sterile syringe fitted with a sterile cannula, 7 ml of 10% glycerol in demineralized water is added to the slant and small bits of growth are scraped from the slant. One milliliter of this heavy suspension is dispensed into each of 6 ampules (T. C. 3D. R. Morris and L. P. Hager,J. Biol. Chem. 241, 1763 (1966).
[84]
CHLOROPEROXIDASE
651
Wheaton No. 12483) and sealed. The sealed ampules are then placed in a Linde BF-5 Biological Freezer and adjusted to a cooling rate of 1° per minute. The frozen ampules are held in a liquid nitrogen refrigerator. When an ampule is needed, one is removed from the rack, placed in a beaker of 40 ° water and agitated until it has thawed. The vial is opened and the contents are planted on a potato-dextrose agar slant. Inoculum for flasks is routinely prepared by seeding potato-dextrose agar plates from slant cultures. Two different colony types can be distinguished on the plates. The colony type which gives high chloroperoxidase-yielding cultures is black, moist, and bristly. The other colony type is greenish gray and has a velvety texture. After 2-3 weeks of growth at room temperature, plates are stored at 4 ° until needed for inoculation. One large colony serves as the inoculum for a 600-ml growth flask. Purification P r o c e d u r e All steps were carried out in the cold room at 4 °.
Step 1. Chromatography on Calcium Phosphate Gel-Cellulose. When chloroperoxidase excretion reaches a peak, 4 growth flasks are pooled and the mycelium is removed by filtration on a Biichner funnel using a No. 44 nylon monofilament (obtained from Tobler, Ernst and Traber Co., New York). The filtrate (approximately 2.3 liters) is dialyzed overnight 3 successive times using 40 liters of 0.01 M potassium phosphate buffer, pH 5.0, for each dialysis. The dialyzate is centrifuged at 16,300 g for 1 hour to remove particulate material. The clear amber-colored supernatant fraction (approximately 2.8 liters) is charged on a 7 × 7 cm calcium phosphate gel-cellulose column, prepared according to the method described by Swingle and Tiselius. 4 Chloroperoxidase adsorbs to the column in a black band at the top. The column is first washed with 200 ml of 0.01 M potassium phosphate buffer, pH 5. Chloroperoxidase is then eluted from the column with 1 liter of 0.2 M potassium phosphate buffer, pH 5.0. Fifteen milliliter fractions are collected. Step 2. Crystallization. The 10 peak fractions from the calcium phosphate gel-cellulose column are combined and concentrated by dialysis for 6 hours against 30 volumes of saturated ammonium sulfate. Precipitated protein is removed by centrifugation at 34,000 g for 30 minutes. T h e precipitate is redissolved in an amount (approximately 6 ml) of 0.01 M potassium phosphate buffer to yield a protein concentration of 5-10 mg/ml. The concentrated fractions are dialyzed overnight against 100 volumes of 60% saturated ammonium sulfate. The 4S. M. Swingle and H. Tiselius,
Biochem.J.48, 171 ( 1951).
652
[84]
AROMATIC AMINO ACIDS
enzyme preparation is removed from the dialysis bag, and saturated ammonium sulfate is added dropwise to a final concentration of 70% saturation. The sample is placed at 4 °. Crystallization usually begins within 6 to 24 hours. The results of a typical purification are shown in the table. PURIFICATION OF CHLOROPEROXIDASE FROM
Fraction
Volume (ml)
Crude extract, dialyzed Centrifuged supernatant Calcium phosphatecellulose eluate First crystallization Second crystallization Third crystallization
Caldariomycesfumago
Total activity (p.moles/ min X 10 -4)
Total protein (mg)
Specific activity (g.moles/ min/mg)
24.8 24.4 12.4
12000 6200 94.2
20 39 1316
55.0 26.8 13.1
1480 1660 1600
2920 2880 150 6.5 6.5 3.5
8.15 4.45 2.10
Properties
Spectrophotometric Properties of Chloroperoxidase. The spectrum of native chloroperoxidase has absorption maxima at 403, 515, 542, and 650 m/.~3. The millimolar extinction coefficients are, respectively, 75.3, 11.5, 10.8, and 4.2. Physical Properties of Chloroperoxidase. The sedimentation constant (s20,w) of chloroperoxidase, based on an extrapolated value to zero protein concentration, is 4.1 S? The minimal molecular weight, based on a dry weight vs. heme content (determined by pyridine hemochromogen) is 42,000. Chemical Properties. The molecular weight, on the basis of amino acid composition (plus heme) is 28,640. The remaining 13,000-14,000 molecular weight equivalents can be accounted for as carbohydrate. The principal monomeric components of the carbohydrate portion of the enzyme have been identified as mannose, glucose, and galactose in a ratio of 2:1 : 1.5
SR. Glew and E. Heath, personal communication.
AROMATIC AMINO ACIDS
[84]
pH Optimum. The pH optimum for the enzyme in 0.05 M Tris buffer is 8.2.
Specificity. The enzyme oxygenates 3,4-dihydroxyphenylacetic acid (1), ~ 3,4-dihydroxyphenylpropionic acid (1/35), 7 3,4-dihydroxybenzoic acid (1/1,200), * and catechol (1/18,000), 7 but does not oxygenate dopamine. Activators and Inhibitors. The enzyme is a simple protein which contains 5 atoms of iron as the sole prosthetic group and 16 residues of sulfhydryl groups per mole of the enzyme. Fe 2+ serves as an activator to enzyme preparations inactivated either by prolonged storage under air or treatment with hydrogen peroxide, and as a stabilizer to the native enzyme. The participation of the enzyme bound iron in the reaction, and its valency changes from ferrous to ferric are demonstrated by the ESR studies of the reaction, s The enzyme is inhibited competitively by 0-phenanthroline and 8-hydroxyquinoline, and noncompetitively by Hg 2+ and PCMB. The treatment of the enzyme with 8 equivalents of PCMB causes the dissociation of the enzyme into 4 subunits, which are inactive. 7Numbers in parentheses represent relative rates of oxidation. SH. Kita, M. Kamimoto, and S. Senoh, 7th Intern. Congr. Biochem., Tokyo, Abstr. IV-F-87 (1967).
[84] Iodination of Tyrosine by Chloroperoxidase; Preparation of Chloroperoxidase (Caldariomyces fumago) By LOWELL P. HAGER x - + H202 + H-acceptor --* X-acceptor + O H - + HzO
(1)
Chloroperoxidase catalyzes peroxidative halogenation reactions, with chloride, bromide, and iodide ions serving as the halogen anion donor. TM The reaction is relatively nonspecific with respect to the acceptor molecule. Any good nucleophile appears to possess acceptor activity; /3-keto acids and/3-diketones, by virtue of having an enolizable proton, are extremely active acceptors. Halide ions, especially iodide ion, are also good acceptors. Thus, under appropriate conditions, chloroperoxi1See articles [85a] and [85b] for a description of the iodination of tyrosine catalyzed by bovine lactoperoxidase and pig thyroid peroxidase. laL. P. Hager, D. R. Morris, F. S. Brown, and H. Eberwein,J. Biol. Chem. 241, 1769 (1966).
[84]
CHLOROPEROXIDASE
649
dase will catalyze the peroxidative formation of molecular iodine, bromine, and chlorine according to Eq. (2). 2x-~- H202 ---, X2 + 2OH-
(2)
Aromatic compounds possessing enolic character also serve as acceptors 2 although the overall rate of the halogenation reaction is variable and severalfold lower, with tyrosine, anisole, and benzyl alcohol serving as acceptors in decreasing order of reactivity. In the special case when iodide serves as the halogen donor and tyrosine serves as the halogen acceptor, it can be readily shown that molecular iodine is an obligate intermediate in the reaction. This follows from the fact that iodide ion is a much better acceptor in Eq. (1) than is tyrosine. Thus, the initial peroxidation of iodide ion produces elemental iodine. The subsequent utilization of molecular iodine leads to tyrosine iodination according to Eq. (3). Iz + H202 + tyrosine ~
2 monoiodotyrosine + H20
(3)
Crystalline chloroperoxidase preparations catalyze both reactions illustrated in Eqs. 2 and 3. Chloroperoxidase, a glycoprotein, is excreted in large amounts by Caldariomycesfumago in its phase of secondary metabolism. After an initial growth period in shake flasks of 6-7 days, chloroperoxidase excretion commences and continues for a 3-4-day period. Chloroperoxidase concentrations in the culture medium reach levels as high as 100 mg per liter of culture medium. Assay Method
The tyrosine iodination assay method for chloroperoxidase is based on the loss of tyrosine fluorescence in the conversion of tyrosine to its mol,oiodo derivative. The complete reaction mixture contains 300 micromoles of potassium phosphate buffer, pH 2.75, za 1 micromole o f tyrosine, 1.5 micromoles of hydrogen peroxide, 1 micromole of potassium iodide, and enzyme in total volume of 3 ml. The reaction is initiated by the addition of enzyme. Tyrosine fluorescence is measured at 303 m/.~ after irradiation at 275 m/~. There is an initial rapid decrease in fluorescence yield which is due to the formation of molecular iodine, which acts as screen and decreases the 275 m/~ irradiation. Subsequent utilization of the molecular iodine for the iodination of tyrosine yields 2F. S. Brown and L. P. Hager, MeetingAm. Chem. Soc., New York (1966). 2aThe pH 2.75 buffer is prepared by mixing 0.1 M HaPO4 and 0.1 M KH2PO4. The nonenzymatic iodination of tyrosine by 12 at pH 2.75 is so slow that it is of no quantitative significance.
650
AROMATIC AMINO ACIDS
[84]
a decrease in fluorescence that is proportional to the enzyme concentration. Routine assay of chloroperoxidase can be more easily accomplished in a reaction that measures the rate of halogenation of monochlorodimedone. The monochlorodimedone assay3 for chloroperoxidase is based on the loss of absorbance at 278 m/z that accompanies the conversion of 1,1-dimethyl-4-chloro-3,5-cyclohexanedione (monochlorodimedone) to 1,1-dimethyl-4,4-dichloro-3,5-cyclohexanedione (dichlorodimedone). The assay mixture contains 300 micromoles of potassium phosphate buffer, pH 2.75, 60 micromoles of potassium chloride, 6 micromoles of hydrogen peroxide, 0.3 micromole of monochlorodimedone, and a suitable aliquot of enzyme in a total volume of 3 ml. The reaction is started by the addition of enzyme. The change in absorbance at 278 m/J, as a function of time is recorded. The rate of the reaction is linear for 3-4 minutes. One unit of chloroperoxidase activity has been defined as the formation of 1 micromole of dichlorodimedone per minute under these assay conditions. Since the molar extinction coefficient of monochlorodimedone at 278 m/~ is 1.22 x 104, and dichlorodimedone has negligible absorption at this wavelength, the absorbancy change per minute times 0.246 is equal to 1 enzyme unit. Specific activity is defined as units per milligram of protein.
Preparation of the Enzyme Growth Medium. T h e mold is grown in a medium containing 4% glucose, 2% malt extract, 0.2% NaNO3, 0.2% KCI, 0.2% KH2PO4; 0.1% MgSO4" 7HzO, and 0.002% FeSO4.7H~O. One-liter Erlenmeyer flasks containing 600 ml of growth medium are incubated at 23-24 ° on a rotary shaker (New Brunswick, Model V) at 240 rpm. Chloroperoxidase excretion commences after approximately 7 days' incubation. Enzyme production usually reaches a maximum after 10-12 days' incubation. Chloroperoxidase excretion usually falls between the limits of 20 and 100 mg enzyme per liter of culture medium. lnoculum. Caldariomycesfumago (ATCC 16373) is maintained at liquid nitrogen temperature as a mycelial suspension frozen in 10% glycerolwater. The following procedure has been developed by Mrs. Frances A r n o w , Curator, Squibb Culture Collection, for stock culture maintenance. A slant of C. fumago is prepared on potato-dextrose agar and incubated 10-14 days at 25 °. With a sterile syringe fitted with a sterile cannula, 7 ml of 10% glycerol in demineralized water is added to the slant and small bits of growth are scraped from the slant. One milliliter of this heavy suspension is dispensed into each of 6 ampules (T. C. 3D. R. Morris and L. P. Hager,J. Biol. Chem. 241, 1763 (1966).
[84]
CHLOROPEROXIDASE
651
Wheaton No. 12483) and sealed. The sealed ampules are then placed in a Linde BF-5 Biological Freezer and adjusted to a cooling rate of 1° per minute. The frozen ampules are held in a liquid nitrogen refrigerator. When an ampule is needed, one is removed from the rack, placed in a beaker of 40 ° water and agitated until it has thawed. The vial is opened and the contents are planted on a potato-dextrose agar slant. Inoculum for flasks is routinely prepared by seeding potato-dextrose agar plates from slant cultures. Two different colony types can be distinguished on the plates. The colony type which gives high chloroperoxidase-yielding cultures is black, moist, and bristly. The other colony type is greenish gray and has a velvety texture. After 2-3 weeks of growth at room temperature, plates are stored at 4 ° until needed for inoculation. One large colony serves as the inoculum for a 600-ml growth flask. Purification P r o c e d u r e All steps were carried out in the cold room at 4 °.
Step 1. Chromatography on Calcium Phosphate Gel-Cellulose. When chloroperoxidase excretion reaches a peak, 4 growth flasks are pooled and the mycelium is removed by filtration on a Biichner funnel using a No. 44 nylon monofilament (obtained from Tobler, Ernst and Traber Co., New York). The filtrate (approximately 2.3 liters) is dialyzed overnight 3 successive times using 40 liters of 0.01 M potassium phosphate buffer, pH 5.0, for each dialysis. The dialyzate is centrifuged at 16,300 g for 1 hour to remove particulate material. The clear amber-colored supernatant fraction (approximately 2.8 liters) is charged on a 7 × 7 cm calcium phosphate gel-cellulose column, prepared according to the method described by Swingle and Tiselius. 4 Chloroperoxidase adsorbs to the column in a black band at the top. The column is first washed with 200 ml of 0.01 M potassium phosphate buffer, pH 5. Chloroperoxidase is then eluted from the column with 1 liter of 0.2 M potassium phosphate buffer, pH 5.0. Fifteen milliliter fractions are collected. Step 2. Crystallization. The 10 peak fractions from the calcium phosphate gel-cellulose column are combined and concentrated by dialysis for 6 hours against 30 volumes of saturated ammonium sulfate. Precipitated protein is removed by centrifugation at 34,000 g for 30 minutes. T h e precipitate is redissolved in an amount (approximately 6 ml) of 0.01 M potassium phosphate buffer to yield a protein concentration of 5-10 mg/ml. The concentrated fractions are dialyzed overnight against 100 volumes of 60% saturated ammonium sulfate. The 4S. M. Swingle and H. Tiselius,
Biochem.J.48, 171 ( 1951).
652
[84]
AROMATIC AMINO ACIDS
enzyme preparation is removed from the dialysis bag, and saturated ammonium sulfate is added dropwise to a final concentration of 70% saturation. The sample is placed at 4 °. Crystallization usually begins within 6 to 24 hours. The results of a typical purification are shown in the table. PURIFICATION OF CHLOROPEROXIDASE FROM
Fraction
Volume (ml)
Crude extract, dialyzed Centrifuged supernatant Calcium phosphatecellulose eluate First crystallization Second crystallization Third crystallization
Caldariomycesfumago
Total activity (p.moles/ min X 10 -4)
Total protein (mg)
Specific activity (g.moles/ min/mg)
24.8 24.4 12.4
12000 6200 94.2
20 39 1316
55.0 26.8 13.1
1480 1660 1600
2920 2880 150 6.5 6.5 3.5
8.15 4.45 2.10
Properties
Spectrophotometric Properties of Chloroperoxidase. The spectrum of native chloroperoxidase has absorption maxima at 403, 515, 542, and 650 m/.~3. The millimolar extinction coefficients are, respectively, 75.3, 11.5, 10.8, and 4.2. Physical Properties of Chloroperoxidase. The sedimentation constant (s20,w) of chloroperoxidase, based on an extrapolated value to zero protein concentration, is 4.1 S? The minimal molecular weight, based on a dry weight vs. heme content (determined by pyridine hemochromogen) is 42,000. Chemical Properties. The molecular weight, on the basis of amino acid composition (plus heme) is 28,640. The remaining 13,000-14,000 molecular weight equivalents can be accounted for as carbohydrate. The principal monomeric components of the carbohydrate portion of the enzyme have been identified as mannose, glucose, and galactose in a ratio of 2:1 : 1.5
SR. Glew and E. Heath, personal communication.