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Cystathionine γ-lyase from Streptomyces phaeochromoǵenes
486
ENZYMES
[85]
Properties
Stability. Following sterile filtration, the enzyme was stable when stored at 4 ° for up to 4 months. The enzyme lost activity when frozen or lyophilized but remained stable at -20 ° in the presence of 50% glycerol for 4 months. Unless grown in the presence of colicin, the plasmid may be unstable, resulting in a loss of the overproduction phenotype. pH Optimum. The enzyme showed a bell-shaped pH-rate profile with an optimum between pH 8 and 9. At pH 7.2 the enzyme had 36% of its optimal activity. Substrate Specificity. The enzyme followed Michaelis-Menten kinetics and showed broad substrate specificity. Table II presents several kinetic properties. Using the coupled enzyme assay 3 in the presence of 10 mM dithioerythritol, L-cysteine was a poor substrate with a maximal velocity only 1% of that with L-cystathionine. L-Cysteine also showed excess substrate inhibition. Inhibitors. Adenosine (1 mM), S-adenosylmethionine (1 raM), Sadenosylhomocysteine (1 raM), allylglycine (16 mM), diaminopimelic acid (16 mM), 2,7-diaminooct-4-ynedioic acid (16 mM), and propargylglycine (1 raM) did not significantly inhibit the enzyme, fl-Cyanoalanine inhibited to only 43% at 16 mM. L-Cysteine showed competitive inhibition with a K~ of 0.4 mM in the presence of l0 mM dithioerythritol. 3,3,3-Trifluoroalanine showed time-dependent irreversible inhibition with a binding constant of 0.55 mM and a saturating inactivation half-time of 1.7 min. Physical Properties. The enzyme had an aggregate size of 280,000 as determined by gel filtration, with 6 subunits of 45,000 determined by sodium dodecyl sulfate gel electrophoresis. It bound 6 tool of pyridoxal phosphate with a Km of 5/zM. All 36 cysteine residues could react with DTNB, indicating that there were no disulfide bonds.
[85] C y s t a t h i o n i n e y - L y a s e f r o m
Streptomyces phaeochromogenes B y T O R U N A G A S A W A , H I R O S H 1 K A N Z A K I , and H I D E A K I Y A M A D A L-Cystathionine + H20 ~
L-cysteine + H20 + a - k e t o b u t y r i c acid
Cystathionine y-lyase (EC 4.4.1.1) was first purified and crystallized from rat liver, 1 but is also widely distributed among fungi. It has been ~ Y. Matsuo and D. M. Greenberg, J. Biol. Chem. 230, 545 (1958).
METHODS IN ENZYMOLOGY, VOL. 143
Copyright (~ 1987by AcademicPress, Inc. All rightsof reproduction in any form reserved.
[85]
CYSTATHIONINE~'-LYASE
487
shown to be present in S a c c h a r o m y c e s , 2"3 Neurospora, 2 and Aspergillus. 4 Although previous studies all indicated that prokaryotic organisms lack cystathionine y-lyase activity, 2'5 the e n z y m e is now known to be widely distributed in actinomycetes: Streptomyces, Micromonospora, Micropolyspora, Mycohacterium, Nocardia, Streptosporangium, and Streptouerticillium. 6 Strains belonging to the genus S t r e p t o m y c e s show relatively strong cystathionine -,/-lyasc activity. The microbial enzyme has been purified and crystallized from S t r e p t o m y c e s p h a e o c h r o m o g e n e s (IFO 3105). 6 Cystathionine/3-synthase and cystathionine y-synthasc activities were also detected in crude extracts of S. p h a e o c h r o m o g e n e s , but cystathionine/3-1yase was not. Consequently, the reverse transsulfuration pathway in actinomycetcs appears to be similar to that in yeast and molds. 6 Assay Procedures Principle. The e n z y m e is assayed by measuring the rate of formation of either o~-ketobutyric acid or cysteine from L-cystathionine, or the formation of a-ketobutyric acid from L-homoserine. Gaitonde's acid/ninhydrin assay v is highly specific, there being essentially no reaction with homocysteine. The lack of color development with homocysteine means that the assay is specific for y-lyase activity and that/3-lyase activity does not interfere. The assay is suitable, therefore, for crude extracts with cystathionine as substrate. A s s a y 1: M e a s u r e m e n t o f a-Ketobutyric A c i d F o r m e d .fi'om t,-Homoserine Reagents
Potassium phosphate, 0.2 M, pH 8.0 L-Homoserine, 0.2 M Pyridoxal phosphate, I mM Trichloroacetic acid, 1.84 M Procedure. To a centrifuge tube are added 0.2 ml potassium phosphate, 0.25 ml L-homoserine, 40 ~1 pyridoxal phosphate, 0. I-0.5 units of 2 C. Delavier-Klutchko and M. Flavin, J. Biol. Chem. 240, 2537 (1965). 3 E. Morzycka and A. Paszewski, FEBS Lett. 101, 97 (1979). 4 A. Paszewski and J. Grabski, Acta Biochim. PoL 20, 159 (1973). 5 M. Flavin, in "Metabolic Pathways (D. M. Greenberg, ed.), Vol. 7, p. 457. Academic Press, New York, 1975. T. Nagasawa, H. Kanzaki, and H. Yamada, J. Biol. Chem. 259, 10393 (19841. 7 M. Gaitonde, Biochern. J. 104, 626 (1967).
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ENZYMES
[85]
enzyme, and water to a total volume of 1.0 ml. For each assay tube, a control tube is prepared from which c-homoserine is omitted. The mixture is incubated for 30 min at 30°; during this period a linear rate should be attained. Incubation is terminated by adding 0.2 ml of trichloroacetic acid. The precipitate is removed by centrifugation, and a-ketobutyric acid in the supernatant fluid is determined by the method of Friedemann and Haugen. 8
Assay 11: Measurement of t~-Cysteine Formed from l~-Cystathionine Reagents Potassium phosphate, 0.2 M, pH 8.0 L-Cystathionine, 8.3 mM Pyridoxal phosphate, I mM Trichloroacetic acid, 1.84 M Procedure. The reaction is carried out as described for Assay I except that the L-homoserine is replaced by 0.25 ml of L-cystathionine. The incubation is stopped by adding 0.2 ml of trichloroacetic acid. The precipitate is removed by centrifugation, and cysteine is determined by the method of Gaitonde. 7 Definition of Unit and Specific Activity. One unit of enzyme is defined as the amount of enzyme catalyzing the formation of 1/~mol of a-ketobutyric acid from L-homoserine in 1 min. Specific activity is expressed in terms of enzyme units per milligram protein. Protein is determined from its absorption at 280 nm. The absorption coefficient was found to be 0.839 mg -t ml cm t by absorbance and by dry weight determination. 6 Purification Procedure
Source and Growth. Streptomyces phaeochromogenes (IFO 3105) obtained from a stock culture of the Institute of Fermentation, Osaka (IFO Type Culture Collection), was the source of the enzyme. The basal medium for cultivation consists of 2 g yeast extract, 0.3 g glucose, 0.2 g K2HPO4, 0.1 g MgCI2" 2H20, 4 mg CaCI2" 2H20, 2 mg FeSO4-7H20, 2 mg MnSO4.4-6H20, 2 mg CuSO4" 5H20, and I mg ZnSO4.7H20 per 100 ml of tap water. The pH of the medium is adjusted to 7.0 with 4 M NaOH. Streptomyces phaeochromogenes is collected from an agar slant of the basal medium and inoculated into subculture flask. The subculture (3.5 liters) is shaken reciprocally at 28 ° for 48 hr and then inoculated into a 100liter jar fermentor containing 70 liters of the basal medium supplemented s T. E. Friedemann, this series, Vol. 3, p. 414.
[85]
CYSTATHIONINE y-LYASE
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with 35 g of antifoam (AF-emulsion). Incubation is carried out at 28° for 20 hr with aeration (37 liters/min). The cells from 33.5 liters of broth are harvested by continuous-flow centrifugation and washed with 0.15 M NaC1 containing 0.1 mM EDTA. The yield of wet cells is approximately 20 g/liter of medium. All operations in steps 1 through 9 are carried out at 4°, and potassium phosphate buffer containing 20 p,M pyridoxal phosphate, 0.12 mM EDTA, and 0.2 mM dithiothreitol is used throughout, unless otherwise specified. Step 1: Cell-Free Extract. Washed cells (670 g) are suspended in 2 liters of 0.1 M potassium phosphate at pH 7.5 and disrupted by ultrasonic oscillation (19 kHz). Cell debris is removed by centrifugation at 12,000 g for 30 min. The supernatant fluid is dialyzed overnight against 10 mM potassium phosphate at pH 7.5 at 5°. The precipitate that appears during dialysis is removed by centrifugation at 12,000 g for 30 min and discarded. Step 2: Salt Fractionation I. Solid ammonium sulfate, 17.6 g, is added per I00 ml of cell-free extract (30% saturation) at 0°. The pH is maintained at 7.5 with 6.3 M NH4OH. After stirring for 2 hr or more, the precipitate is removed by centrifugation at 12,000 g, and 19.8 g of ammonium sulfate is added per 100 ml of fluid (60% saturation). The suspension is centrifuged at 12,000 g, and the pellet dissolved in 0.1 M potassium phosphate at pH 7.5. The solution is dialyzed for 36 hr against three changes of 10 liters each of 10 mM potassium phosphate at pH 7.5. Step 3: DEAE-Cellulo6"e. The enzyme solution is applied to a DEAEcellulose column (3.5 x 60 cm), previously equilibrated with 0.1 M potassium phosphate at pH 7.5. The column is washed with 0.1 M potassium phosphate buffer (pH 7.5) containing 0.1 M KCI until the absorbance of the effluent at 280 nm is reduced to 0.2 or less. The enzyme is eluted with the same buffer containing 0.2 M KC1. Fractions of 17 ml are collected, the active eluates are combined, and 39 g of solid ammonium sulfate are added per 100 ml with stirring. The precipitate is collected by centrifugation, dissolved in a small amount of the phosphate buffer, and dialyzed against three changes of 10 liters each of 10 mM potassium phosphate at pH 7.5. Step 4: Salt Fractionation H. The dialyzed enzyme preparation from Step 3 is again subjected to fractionations with ammonium sulfate (finely ground). The precipitate appearing between 50 and 60% of saturation (31.1 g and 39 g per 100 ml, respectively) is dissolved in the 0.1 M phosphate buffer and dialyzed against three changes of 5 liters each of the 10 mM phosphate buffer. Step 5: DEAE-Sephacel. The enzyme solution from Step 4 is applied to a column (2.7 x 27 cm) of DEAE-Sephacel previously equilibrated with
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ENZYMES
[85]
0.1 M phosphate buffer. After the column has been washed with the same buffer, the enzyme is eluted with a linear gradient of KCI (0-0.5 M, 400 mi in each container) in the same buffer at a flow rate of 80 ml/hr. Fractions of 3.3 ml are collected, and the active fractions are pooled. Solid ammonium sulfate is added to the enzyme solution to 39 g/100 ml of solution. After centrifugation of the suspension at 12,000 g, the precipitate is dissolved in the 0. l M buffer and dialyzed against 5 liters of 10 mM buffer. Step 6: Hydroxylapatite. The enzyme solution is applied to a column (2.8 x 14 cm) of hydroxylapatite previously equilibrated with the 10 mM phosphate buffer. The column is washed fully with l0 mM phosphate until the absorbance of the effluent at 280 nm is reduced to 0.1 or less. The enzyme is then eluted with 200 ml of 50 mM potassium phosphate. Fractions of 3 ml are collected, and the active fractions are combined. Step 7: Phenyl-Sepharose CL-4B. After cooling to 0°, 7.67 g/100 ml of ammonium sulfate is added in small portions with stirring (10% of saturation). The enzyme solution is applied to a column (1.0 x 26 cm) of phenylSepharose CL-4B previously equilibrated with 50 mM potassium phosphate at pH 7.5 containing 10% saturated ammonium sulfate. The column is washed with 50 ml of 50 mM potassium phosphate at pH 7.5 containing sequentially 10% and 5% saturated ammonium sulfate. The enzyme is eluted by lowering linearly the ionic strength of ammonium sulfate (from 5 to 0%, 200 ml in each container) in the same buffer. Fractions of 2.3 ml are collected, and the active fractions are combined. Step 8: Octyl-Sepharose CL-4B. Ammonium sulfate is added in small portions with stirring to bring the enzyme solution from Step 7 to 10% saturation (5.6 g/100 ml). After application to a column (0.8 x 10 cm) of octyl-Sepharose CL-4B, previously equilibrated with 50 mM phosphate buffer containing 10% ammonium sulfate, the enzyme passes through the column without loss of activity. Step 9: Sephadex G-200. Ammonium sulfate, 32.6 g/100 ml, is added in small portions with stirring to the solution from Step 8. The precipitate is collected at 12,000 g and dissolved in 10 mM potassium phosphate buffer. The enzyme solution is concentrated to about 2 ml with a Diaflo YM30 membrane and charged onto a column (2.3 x 93 cm) of Sephadex G-200 previously equilibrated with 10 mM potassium phosphate at pH 7.5 containing 0.1 M KCl. The rate of sample loading and column elution is maintained at 4 ml/hr with a peristaltic pump. Protein is eluted with the same buffer, and fractions containing cystathionine y-lyase activity are combined and concentrated by uitrafiltration. Purification of approximately 3,000-fold can be achieved, with a yield of about 30%. After the last step, the enzyme appears to be homogeneous
[85]
CYSTATHION1NE 7-LYASE
491
TABLE I PURIFICATION OF CYSTATHIONINE 'y-LYASE FROM S. phaeochrornogenes"
Step I. 2. 3. 4. 5. 6. 7. 8. 9.
Cell-free extract Salt fractionation I DEAE-Cellulose Salt fractionation II DEAE-Sephacel Hydroxylapatite Phenyl-Sepharose CL-4B Octyl-Sepharose CL-4B Sephadex G-200
Total volume (ml)
Total protein (rag)
Total activity (units)
Specific activity (units/rag)
2,210 800 435 43.5 106 64.8 9.5 18 48.2
85,800 48,500 1,970 582 279 114 21.3 9.54 8.15
73.3 70.6 69.7 43.2 41.2 32.1 22.3 21.8 21.2
0.0009 0.0015 0.0483 0.0741 0.148 0.383 1.05 2.28 2.60
,' The reaction was carried out under the conditions of Assay I1. by the criteria of p o l y a c r y l a m i d e gel electrophoresis, analytical centrifugation, and double diffusion in agarose. The purified e n z y m e catalyzes the a,y-elimination reaction of L-homoserine (Assay l) and e-cystathionine (Assay I1) at 2.60 and 1.90 p,mol/min/ mg of protein, respectively. The purification procedure is summarized in Table I. Crystallization. The purified e n z y m e solution is diluted with l0 m M of the p o t a s s i u m p h o s p h a t e buffer containing 20 /zM pyridoxal phosphate and 0.2 m M dithiothreitol to a protein concentration of about 30 mg/ml. A m m o n i u m sulfate is added to this solution to 31.3 g/100 mi at 0 ° with gentle stirring with a glass rod. Additional a m m o n i u m sulfate is added until the induced turbidity ceases to disappear upon stirring. The presence of cystallized materials is evident from the silky sheen seen when the mixture is stirred. I f denatured protein strands b e c o m e visible, the solution is centrifuged to r e m o v e them before the e n z y m e begins to crystallize. The cystallized e n z y m e is in the apo form. Full activity is restored by the addition of 2 0 / z M pyridoxal phosphate. Properties
Stability. The highly purified e n z y m e can be stored for 2 weeks without loss of activity in 10 m M potassium phosphate at pH 7.5 containing 20 /~M pyridoxal p h o s p h a t e , 0.12 m M E D T A , and 0.2 m M dithiothreitol at 4 °. The e n z y m e remains stable for 2 or more months at - 2 0 ° in the a b o v e buffer containing 45% (v/v) glycerol.
492
ENZYMES
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pH Optimum. In two buffer systems, Tris-HCl and NH4CI-NH4OH (final concentration, 42 raM), the optima for the a,y-elimination reaction of L-cystathionine and L-homoserine are at approximately pH 9.0. Mr and Cofactor. The enzyme has an Mr of about 166,000 and consists of four subunits of identical size. The holoenzyme exhibits absorption maxima at 278 and 421 nm with a n A278 : A421 ratio of approximately 3.97, and contains 4 mol of pyridoxal phosphate per mole of enzyme. Substrate Specificity and Catalytic and Kinetic Properties. L-Cystathionine, L-homoserine, DL-ianthionine, L-djenkolic acid, and L-cystine are cleaved by the Streptomyces enzyme. L-Cysteine is also cleaved by the enzyme, although at concentrations greater than 1 mM, substrate inhibition is seen. For L-cystathionine and c-homoserine the apparent Km values are 0.2 and 13 mM, respectively. The a,fl-elimination reaction of Lcystathionine is also catalyzed by the enzyme at a rate about 15% of that of the a,y-elimination reaction. The enzyme can also catalyze the yreplacement reaction of L-homoserine in the presence of L-cysteine to form L-cystathionine. 9 D-Cysteine, L- and o-homocysteine, and 3- and 2mercaptopropionate can replace L-cysteine as substrates for y-replacement. )o lnhibitors. The enzyme is strongly inhibited by carbonyl reagents such hydroxylamine, l)- and L-penicillamine, semicarbazide, phenylhydrazine, 3-methyl-2-benzothiazolinone hydrazone, and D-cycloserine. The enzyme displays high sensitivity to some thiol reagents (at 1 mM), e.g., pchloromercuribenzoate, HgC12, AgNO3, and ZnCI2, yielding from 60 to 100% inhibition after 30 min at 30° with each reagent. Sodium cyanide causes 82% inhibition at I mM. DL-Propargylglycine and /3-cyano-Lalanine strongly inhibit (80-100% inhibition at I mM). Immunological Properties. Antiserum prepared against the purified cystathionine y-lyase of S. phaeochromogenes cross-reacts with the cystathionine y-lyase from S. olivochromogenes (IFO 3178), S. lydicus (AKU 2502), S. lactamdurans (IFO 13305), Streptoverticillium kentuchense (IFO 12880), Streptosporangium roseum (IFO 3776), Micromonospora chalcea (IFO 12135), Micropolyspora angispora (IFO 13155), and Microellobosporia violacea (IFO 12517). Spurs are formed when the enzymes from these actinomycetes are placed in neighboring wells on double-immunodiffusion analysis.
9 H. Yamada, H. Kanzaki, and T. Nagasawa, J. Biotechnol. 1, 205 (1984). 10 H. Kanzaki, M. Kobayashi, T. Nagasawa, and H. Yamada, Agric. Biol. Chem. 50, 391 (1986).
ENZYMES
[85]
Properties
Stability. Following sterile filtration, the enzyme was stable when stored at 4 ° for up to 4 months. The enzyme lost activity when frozen or lyophilized but remained stable at -20 ° in the presence of 50% glycerol for 4 months. Unless grown in the presence of colicin, the plasmid may be unstable, resulting in a loss of the overproduction phenotype. pH Optimum. The enzyme showed a bell-shaped pH-rate profile with an optimum between pH 8 and 9. At pH 7.2 the enzyme had 36% of its optimal activity. Substrate Specificity. The enzyme followed Michaelis-Menten kinetics and showed broad substrate specificity. Table II presents several kinetic properties. Using the coupled enzyme assay 3 in the presence of 10 mM dithioerythritol, L-cysteine was a poor substrate with a maximal velocity only 1% of that with L-cystathionine. L-Cysteine also showed excess substrate inhibition. Inhibitors. Adenosine (1 mM), S-adenosylmethionine (1 raM), Sadenosylhomocysteine (1 raM), allylglycine (16 mM), diaminopimelic acid (16 mM), 2,7-diaminooct-4-ynedioic acid (16 mM), and propargylglycine (1 raM) did not significantly inhibit the enzyme, fl-Cyanoalanine inhibited to only 43% at 16 mM. L-Cysteine showed competitive inhibition with a K~ of 0.4 mM in the presence of l0 mM dithioerythritol. 3,3,3-Trifluoroalanine showed time-dependent irreversible inhibition with a binding constant of 0.55 mM and a saturating inactivation half-time of 1.7 min. Physical Properties. The enzyme had an aggregate size of 280,000 as determined by gel filtration, with 6 subunits of 45,000 determined by sodium dodecyl sulfate gel electrophoresis. It bound 6 tool of pyridoxal phosphate with a Km of 5/zM. All 36 cysteine residues could react with DTNB, indicating that there were no disulfide bonds.
[85] C y s t a t h i o n i n e y - L y a s e f r o m
Streptomyces phaeochromogenes B y T O R U N A G A S A W A , H I R O S H 1 K A N Z A K I , and H I D E A K I Y A M A D A L-Cystathionine + H20 ~
L-cysteine + H20 + a - k e t o b u t y r i c acid
Cystathionine y-lyase (EC 4.4.1.1) was first purified and crystallized from rat liver, 1 but is also widely distributed among fungi. It has been ~ Y. Matsuo and D. M. Greenberg, J. Biol. Chem. 230, 545 (1958).
METHODS IN ENZYMOLOGY, VOL. 143
Copyright (~ 1987by AcademicPress, Inc. All rightsof reproduction in any form reserved.
[85]
CYSTATHIONINE~'-LYASE
487
shown to be present in S a c c h a r o m y c e s , 2"3 Neurospora, 2 and Aspergillus. 4 Although previous studies all indicated that prokaryotic organisms lack cystathionine y-lyase activity, 2'5 the e n z y m e is now known to be widely distributed in actinomycetes: Streptomyces, Micromonospora, Micropolyspora, Mycohacterium, Nocardia, Streptosporangium, and Streptouerticillium. 6 Strains belonging to the genus S t r e p t o m y c e s show relatively strong cystathionine -,/-lyasc activity. The microbial enzyme has been purified and crystallized from S t r e p t o m y c e s p h a e o c h r o m o g e n e s (IFO 3105). 6 Cystathionine/3-synthase and cystathionine y-synthasc activities were also detected in crude extracts of S. p h a e o c h r o m o g e n e s , but cystathionine/3-1yase was not. Consequently, the reverse transsulfuration pathway in actinomycetcs appears to be similar to that in yeast and molds. 6 Assay Procedures Principle. The e n z y m e is assayed by measuring the rate of formation of either o~-ketobutyric acid or cysteine from L-cystathionine, or the formation of a-ketobutyric acid from L-homoserine. Gaitonde's acid/ninhydrin assay v is highly specific, there being essentially no reaction with homocysteine. The lack of color development with homocysteine means that the assay is specific for y-lyase activity and that/3-lyase activity does not interfere. The assay is suitable, therefore, for crude extracts with cystathionine as substrate. A s s a y 1: M e a s u r e m e n t o f a-Ketobutyric A c i d F o r m e d .fi'om t,-Homoserine Reagents
Potassium phosphate, 0.2 M, pH 8.0 L-Homoserine, 0.2 M Pyridoxal phosphate, I mM Trichloroacetic acid, 1.84 M Procedure. To a centrifuge tube are added 0.2 ml potassium phosphate, 0.25 ml L-homoserine, 40 ~1 pyridoxal phosphate, 0. I-0.5 units of 2 C. Delavier-Klutchko and M. Flavin, J. Biol. Chem. 240, 2537 (1965). 3 E. Morzycka and A. Paszewski, FEBS Lett. 101, 97 (1979). 4 A. Paszewski and J. Grabski, Acta Biochim. PoL 20, 159 (1973). 5 M. Flavin, in "Metabolic Pathways (D. M. Greenberg, ed.), Vol. 7, p. 457. Academic Press, New York, 1975. T. Nagasawa, H. Kanzaki, and H. Yamada, J. Biol. Chem. 259, 10393 (19841. 7 M. Gaitonde, Biochern. J. 104, 626 (1967).
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ENZYMES
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enzyme, and water to a total volume of 1.0 ml. For each assay tube, a control tube is prepared from which c-homoserine is omitted. The mixture is incubated for 30 min at 30°; during this period a linear rate should be attained. Incubation is terminated by adding 0.2 ml of trichloroacetic acid. The precipitate is removed by centrifugation, and a-ketobutyric acid in the supernatant fluid is determined by the method of Friedemann and Haugen. 8
Assay 11: Measurement of t~-Cysteine Formed from l~-Cystathionine Reagents Potassium phosphate, 0.2 M, pH 8.0 L-Cystathionine, 8.3 mM Pyridoxal phosphate, I mM Trichloroacetic acid, 1.84 M Procedure. The reaction is carried out as described for Assay I except that the L-homoserine is replaced by 0.25 ml of L-cystathionine. The incubation is stopped by adding 0.2 ml of trichloroacetic acid. The precipitate is removed by centrifugation, and cysteine is determined by the method of Gaitonde. 7 Definition of Unit and Specific Activity. One unit of enzyme is defined as the amount of enzyme catalyzing the formation of 1/~mol of a-ketobutyric acid from L-homoserine in 1 min. Specific activity is expressed in terms of enzyme units per milligram protein. Protein is determined from its absorption at 280 nm. The absorption coefficient was found to be 0.839 mg -t ml cm t by absorbance and by dry weight determination. 6 Purification Procedure
Source and Growth. Streptomyces phaeochromogenes (IFO 3105) obtained from a stock culture of the Institute of Fermentation, Osaka (IFO Type Culture Collection), was the source of the enzyme. The basal medium for cultivation consists of 2 g yeast extract, 0.3 g glucose, 0.2 g K2HPO4, 0.1 g MgCI2" 2H20, 4 mg CaCI2" 2H20, 2 mg FeSO4-7H20, 2 mg MnSO4.4-6H20, 2 mg CuSO4" 5H20, and I mg ZnSO4.7H20 per 100 ml of tap water. The pH of the medium is adjusted to 7.0 with 4 M NaOH. Streptomyces phaeochromogenes is collected from an agar slant of the basal medium and inoculated into subculture flask. The subculture (3.5 liters) is shaken reciprocally at 28 ° for 48 hr and then inoculated into a 100liter jar fermentor containing 70 liters of the basal medium supplemented s T. E. Friedemann, this series, Vol. 3, p. 414.
[85]
CYSTATHIONINE y-LYASE
489
with 35 g of antifoam (AF-emulsion). Incubation is carried out at 28° for 20 hr with aeration (37 liters/min). The cells from 33.5 liters of broth are harvested by continuous-flow centrifugation and washed with 0.15 M NaC1 containing 0.1 mM EDTA. The yield of wet cells is approximately 20 g/liter of medium. All operations in steps 1 through 9 are carried out at 4°, and potassium phosphate buffer containing 20 p,M pyridoxal phosphate, 0.12 mM EDTA, and 0.2 mM dithiothreitol is used throughout, unless otherwise specified. Step 1: Cell-Free Extract. Washed cells (670 g) are suspended in 2 liters of 0.1 M potassium phosphate at pH 7.5 and disrupted by ultrasonic oscillation (19 kHz). Cell debris is removed by centrifugation at 12,000 g for 30 min. The supernatant fluid is dialyzed overnight against 10 mM potassium phosphate at pH 7.5 at 5°. The precipitate that appears during dialysis is removed by centrifugation at 12,000 g for 30 min and discarded. Step 2: Salt Fractionation I. Solid ammonium sulfate, 17.6 g, is added per I00 ml of cell-free extract (30% saturation) at 0°. The pH is maintained at 7.5 with 6.3 M NH4OH. After stirring for 2 hr or more, the precipitate is removed by centrifugation at 12,000 g, and 19.8 g of ammonium sulfate is added per 100 ml of fluid (60% saturation). The suspension is centrifuged at 12,000 g, and the pellet dissolved in 0.1 M potassium phosphate at pH 7.5. The solution is dialyzed for 36 hr against three changes of 10 liters each of 10 mM potassium phosphate at pH 7.5. Step 3: DEAE-Cellulo6"e. The enzyme solution is applied to a DEAEcellulose column (3.5 x 60 cm), previously equilibrated with 0.1 M potassium phosphate at pH 7.5. The column is washed with 0.1 M potassium phosphate buffer (pH 7.5) containing 0.1 M KCI until the absorbance of the effluent at 280 nm is reduced to 0.2 or less. The enzyme is eluted with the same buffer containing 0.2 M KC1. Fractions of 17 ml are collected, the active eluates are combined, and 39 g of solid ammonium sulfate are added per 100 ml with stirring. The precipitate is collected by centrifugation, dissolved in a small amount of the phosphate buffer, and dialyzed against three changes of 10 liters each of 10 mM potassium phosphate at pH 7.5. Step 4: Salt Fractionation H. The dialyzed enzyme preparation from Step 3 is again subjected to fractionations with ammonium sulfate (finely ground). The precipitate appearing between 50 and 60% of saturation (31.1 g and 39 g per 100 ml, respectively) is dissolved in the 0.1 M phosphate buffer and dialyzed against three changes of 5 liters each of the 10 mM phosphate buffer. Step 5: DEAE-Sephacel. The enzyme solution from Step 4 is applied to a column (2.7 x 27 cm) of DEAE-Sephacel previously equilibrated with
490
ENZYMES
[85]
0.1 M phosphate buffer. After the column has been washed with the same buffer, the enzyme is eluted with a linear gradient of KCI (0-0.5 M, 400 mi in each container) in the same buffer at a flow rate of 80 ml/hr. Fractions of 3.3 ml are collected, and the active fractions are pooled. Solid ammonium sulfate is added to the enzyme solution to 39 g/100 ml of solution. After centrifugation of the suspension at 12,000 g, the precipitate is dissolved in the 0. l M buffer and dialyzed against 5 liters of 10 mM buffer. Step 6: Hydroxylapatite. The enzyme solution is applied to a column (2.8 x 14 cm) of hydroxylapatite previously equilibrated with the 10 mM phosphate buffer. The column is washed fully with l0 mM phosphate until the absorbance of the effluent at 280 nm is reduced to 0.1 or less. The enzyme is then eluted with 200 ml of 50 mM potassium phosphate. Fractions of 3 ml are collected, and the active fractions are combined. Step 7: Phenyl-Sepharose CL-4B. After cooling to 0°, 7.67 g/100 ml of ammonium sulfate is added in small portions with stirring (10% of saturation). The enzyme solution is applied to a column (1.0 x 26 cm) of phenylSepharose CL-4B previously equilibrated with 50 mM potassium phosphate at pH 7.5 containing 10% saturated ammonium sulfate. The column is washed with 50 ml of 50 mM potassium phosphate at pH 7.5 containing sequentially 10% and 5% saturated ammonium sulfate. The enzyme is eluted by lowering linearly the ionic strength of ammonium sulfate (from 5 to 0%, 200 ml in each container) in the same buffer. Fractions of 2.3 ml are collected, and the active fractions are combined. Step 8: Octyl-Sepharose CL-4B. Ammonium sulfate is added in small portions with stirring to bring the enzyme solution from Step 7 to 10% saturation (5.6 g/100 ml). After application to a column (0.8 x 10 cm) of octyl-Sepharose CL-4B, previously equilibrated with 50 mM phosphate buffer containing 10% ammonium sulfate, the enzyme passes through the column without loss of activity. Step 9: Sephadex G-200. Ammonium sulfate, 32.6 g/100 ml, is added in small portions with stirring to the solution from Step 8. The precipitate is collected at 12,000 g and dissolved in 10 mM potassium phosphate buffer. The enzyme solution is concentrated to about 2 ml with a Diaflo YM30 membrane and charged onto a column (2.3 x 93 cm) of Sephadex G-200 previously equilibrated with 10 mM potassium phosphate at pH 7.5 containing 0.1 M KCl. The rate of sample loading and column elution is maintained at 4 ml/hr with a peristaltic pump. Protein is eluted with the same buffer, and fractions containing cystathionine y-lyase activity are combined and concentrated by uitrafiltration. Purification of approximately 3,000-fold can be achieved, with a yield of about 30%. After the last step, the enzyme appears to be homogeneous
[85]
CYSTATHION1NE 7-LYASE
491
TABLE I PURIFICATION OF CYSTATHIONINE 'y-LYASE FROM S. phaeochrornogenes"
Step I. 2. 3. 4. 5. 6. 7. 8. 9.
Cell-free extract Salt fractionation I DEAE-Cellulose Salt fractionation II DEAE-Sephacel Hydroxylapatite Phenyl-Sepharose CL-4B Octyl-Sepharose CL-4B Sephadex G-200
Total volume (ml)
Total protein (rag)
Total activity (units)
Specific activity (units/rag)
2,210 800 435 43.5 106 64.8 9.5 18 48.2
85,800 48,500 1,970 582 279 114 21.3 9.54 8.15
73.3 70.6 69.7 43.2 41.2 32.1 22.3 21.8 21.2
0.0009 0.0015 0.0483 0.0741 0.148 0.383 1.05 2.28 2.60
,' The reaction was carried out under the conditions of Assay I1. by the criteria of p o l y a c r y l a m i d e gel electrophoresis, analytical centrifugation, and double diffusion in agarose. The purified e n z y m e catalyzes the a,y-elimination reaction of L-homoserine (Assay l) and e-cystathionine (Assay I1) at 2.60 and 1.90 p,mol/min/ mg of protein, respectively. The purification procedure is summarized in Table I. Crystallization. The purified e n z y m e solution is diluted with l0 m M of the p o t a s s i u m p h o s p h a t e buffer containing 20 /zM pyridoxal phosphate and 0.2 m M dithiothreitol to a protein concentration of about 30 mg/ml. A m m o n i u m sulfate is added to this solution to 31.3 g/100 mi at 0 ° with gentle stirring with a glass rod. Additional a m m o n i u m sulfate is added until the induced turbidity ceases to disappear upon stirring. The presence of cystallized materials is evident from the silky sheen seen when the mixture is stirred. I f denatured protein strands b e c o m e visible, the solution is centrifuged to r e m o v e them before the e n z y m e begins to crystallize. The cystallized e n z y m e is in the apo form. Full activity is restored by the addition of 2 0 / z M pyridoxal phosphate. Properties
Stability. The highly purified e n z y m e can be stored for 2 weeks without loss of activity in 10 m M potassium phosphate at pH 7.5 containing 20 /~M pyridoxal p h o s p h a t e , 0.12 m M E D T A , and 0.2 m M dithiothreitol at 4 °. The e n z y m e remains stable for 2 or more months at - 2 0 ° in the a b o v e buffer containing 45% (v/v) glycerol.
492
ENZYMES
[85]
pH Optimum. In two buffer systems, Tris-HCl and NH4CI-NH4OH (final concentration, 42 raM), the optima for the a,y-elimination reaction of L-cystathionine and L-homoserine are at approximately pH 9.0. Mr and Cofactor. The enzyme has an Mr of about 166,000 and consists of four subunits of identical size. The holoenzyme exhibits absorption maxima at 278 and 421 nm with a n A278 : A421 ratio of approximately 3.97, and contains 4 mol of pyridoxal phosphate per mole of enzyme. Substrate Specificity and Catalytic and Kinetic Properties. L-Cystathionine, L-homoserine, DL-ianthionine, L-djenkolic acid, and L-cystine are cleaved by the Streptomyces enzyme. L-Cysteine is also cleaved by the enzyme, although at concentrations greater than 1 mM, substrate inhibition is seen. For L-cystathionine and c-homoserine the apparent Km values are 0.2 and 13 mM, respectively. The a,fl-elimination reaction of Lcystathionine is also catalyzed by the enzyme at a rate about 15% of that of the a,y-elimination reaction. The enzyme can also catalyze the yreplacement reaction of L-homoserine in the presence of L-cysteine to form L-cystathionine. 9 D-Cysteine, L- and o-homocysteine, and 3- and 2mercaptopropionate can replace L-cysteine as substrates for y-replacement. )o lnhibitors. The enzyme is strongly inhibited by carbonyl reagents such hydroxylamine, l)- and L-penicillamine, semicarbazide, phenylhydrazine, 3-methyl-2-benzothiazolinone hydrazone, and D-cycloserine. The enzyme displays high sensitivity to some thiol reagents (at 1 mM), e.g., pchloromercuribenzoate, HgC12, AgNO3, and ZnCI2, yielding from 60 to 100% inhibition after 30 min at 30° with each reagent. Sodium cyanide causes 82% inhibition at I mM. DL-Propargylglycine and /3-cyano-Lalanine strongly inhibit (80-100% inhibition at I mM). Immunological Properties. Antiserum prepared against the purified cystathionine y-lyase of S. phaeochromogenes cross-reacts with the cystathionine y-lyase from S. olivochromogenes (IFO 3178), S. lydicus (AKU 2502), S. lactamdurans (IFO 13305), Streptoverticillium kentuchense (IFO 12880), Streptosporangium roseum (IFO 3776), Micromonospora chalcea (IFO 12135), Micropolyspora angispora (IFO 13155), and Microellobosporia violacea (IFO 12517). Spurs are formed when the enzymes from these actinomycetes are placed in neighboring wells on double-immunodiffusion analysis.
9 H. Yamada, H. Kanzaki, and T. Nagasawa, J. Biotechnol. 1, 205 (1984). 10 H. Kanzaki, M. Kobayashi, T. Nagasawa, and H. Yamada, Agric. Biol. Chem. 50, 391 (1986).