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[106] l -Arabinose isomerase
596
ISOMERASES AND EPIMERASES
[106]
Properties ~
Substrate Specificity. D-Lyxose isomerase has been found only in cells grown on D-lyxose; D-mannose and D-fructose do not serve as inducers. However, the purified enzyme isomerizes D-mannose to D-fructose. The K,~ values for D-lyxose and D-mannose are 3.6 m M and 10 mM, respectively. The Vmax is 2.4 times greater with D-lyxose than with D-mannose. D-Ribose, D- or L-xylose, D- or L-arabinose, D-glucose, D-glucose 6-phosphate, and n-mannose 6-phosphate are not isomerized at a detection level of 0.5% the rate on D-lyxose. Activators and Inhibitors. Activity is nil in the absence of a metal ion. At 1 m M concentrations, the relative activity with various metal salts is as follows: MnC12, 100; FeClD, 78; FeSO~, 18; CaC12, 14; CoCl~, 8; MgC12, 0; ZnS04, 0; CuSO4, 0. The K,~ for Mn ÷÷ is 4 vM. Activity is nil in the presence of 10 ~ / p - c h l o r o m e r c u r i b e n z o a t e ; the activity cannot be restored with the further addition of reduced glutathione, and reduced glutathione was never found to be protective or stimulatory during fractionation or assay. Iodoacetate, oxidized glutathione, 2,6-dichlorophenolindophenol, or KDFe (CN)6 at. 1 m M and pH 7.0 showed no inhibition in the standard assay. Ef]ect of pH. The pH optimum is about 7.0. Activity as a function of pH varies with the buffer, being half maximal at pH 6.3 in maleate and at pH 4.9 in caeodylate. Molecular Weight. The sedimentation coefficient as determined by density gradient centrifugation is 3.7 to 3.8 S, suggesting a molecular weight of about 40,000.
[ 106] L - A r a b i n o s e I s o m e r a s e
By K. YAMANAKAand W. A. WOOD I,-Arabinose ~- L-rihulose This enzyme has been described by Cohen 1 briefly and by Smyrniotis 2 and by Horeekeff '~ in more detail. 1S. S. Cohen, see Vol. I [52]. 2p. Z. Symrniotis, see Vol. V [42]. 8B. L. Horecker, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), p. 178. Academic Press, New York, 1963. E. C. Heath, B. L. Horecker, P. Z. Smyrniotis, and Y. Takagi, J. Biol. Chem. 231, 1031 (1958).
[105]
L-ARABINOSE ISOMERASE
597
Assay Methods 2
Principle. L-Arabinose isomerase activity is assayed speetrophotometrically at 30 ° using a standard spectrophotometer equipped with an absorbancy converter, automatic cuvette positioner, and recorder. ~ The assay system is based on the following sequences of reactions:
L-Arabinose L-Ribulose -t- ATP
L-arabinose isomerase
~L-ribulose
L-ribulokinase
, L-ribulose5-phosphate -{- ADP
Phosphoenolpyruvate ~ ADP Pyruvate ~ NADH
pyruvate kinase
) pyruvate T ATP
lactate dehydrogenase
, lactate ~- NAD
Reagents
Maleate buffer, 0.4 M, pH 6.9 MgC12, 0.1 M ATP, 0.1 M, adjusted to pH 6.9 Phosphoenolpyruvate, 0.025 M, adjusted to pH 6.9 Sodium glutathione NADH, 0.01 M L-Arabinose, 0.1 M Purified L-ribulokinase, free from L-arabinose isomerase6 Lactate dehydrogenase-pyruvate kinase, crystalline (Worthington) All solutions are prepared separately and kept in a deepfreeze. A reagent stock solution is then prepared by mixing 0.8 ml of maleate buffer, 0.4 ml of MgC12, 0.2 ml of ATP, 0.4 ml of phosphoenolpyruvate, 18 mg of glutathione, 0.4 ml of NADH, 0.05 ml of lactate dehydrogenase, and water to a total volume of 4.0 ml. This mixture can be kept frozen and prepared fresh every 3 days. Procedures. To a microcuvette of 0.5-ml capacity and 1-em light path are added 0.1 ml of reagent mixture, 2 units of purified L-ribulokinase, 2 mieromoles of L-arabinose, suitable amounts of L-arabinose isomerase preparation, and water to a volume of 0.15 ml. Blanks are run for NADH oxidase with buffer and NADH, and for ATPase activity by omitting L-ribulokinase and L-arabinose. L-Ribulokinase activity may be determined by the same method by adding L-ribulose instead of L-arabinose. NADH oxidase and ATPase activities are subtracted from the total activity. A linear relationship between the rate of NADH ~W. A. Wood and S. R. Gilford, Anal. Biochem. 2, 589, 601 (1961). 6See this volume [80]. See also F. J. Simpson and W. A. Wood, J. Biol. Chem. 23~), 473 (1958).
598
ISOMERASES AND EPIMERASES
[105]
oxidation and amount of enzyme is obtained between 0 and 0.3 optical density units per minute. This corresponds to 0-0.067 unit of isomerase. Alternate Assay. For crude preparations, the isomerase activity cannot be determined by a spectrophotometric method owing to the strong interference of NADH oxidase and ATPase. In that case, the following procedure is followed: The reaction mixture (1.0 ml) contains 0.5 ml of 0.05M maleate buffer, pH 6.9, for Aerobacter enzyme or of 0.05 M Tris-HCl buffer, pH 8.0, for Lactobacillus enzyme, 0.05 ml of 0.1 M MnCl~ (or MnSO,), and 0.01--0.1 ml of enzyme preparation with adequate dilution. After incubation at 35 ° for 5 minutes, the reaction is started by adding 0.05 ml of 0.1 M L-arabinose, and incubation is continued at 35 ° for 10 minutes. L-Ribulose is determined by the cysteine-carbazole procedures using T,-ribulose-o-nitrophenylhydrazone as a standard. Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount required to effect an absorbancy change of 1.0 per minute at 340 m~ under these conditions. Specific activity is expressed as units per milligram of protein. For the colorimetrie procedure, see Vol. V [42] and the procedure for D-xylose isomerase2 Sources L-Arabinose isomerase activity has been found in the cells grown on T.-arabinose for the following organisms: Lactobacillus plantarum, ",~ some strains of homolaetie acid bacteria related to L. plantarum, 9 and of Aerobacter aerogenes. 1°,11 This enzyme is also produced in L. plantarum grown on D-xylose. ~ However, many of the heterofermentative lactic acid bacteria, such as L. gayonii, L. mannitopous, L. buchneri, L. brevis, L. pen~oaceticus, and L. lycopersici, and some of the Pediococcus group also produce L-arabinose isomerase from the media containing glucose, fructose, galactose, n-xylose, L-arabinose, or malt extract. 9 The glucosegrown cells of L. gayonii contain T,-arabinose isomerase but no L-ribulokinase and NADH oxidase. Induction of L-arabinose isomerase by Pediococcus pentosaceus ~ and by Lactobacillus plantarum ~ was reported. TK. Yamanaka, see this volume [104]. s j. O. Lampen, Proc. Am. Chem. Soe., 44c (1954). 9K. Yamanaka, Bull. Agr. Chem. Soc. Japan 24, 310, (1960) (in English); Mere. Fac. Agr., Kagawa Univ., No. 16 (1963) (in English). •F. J. Simpson, M. J. Wolin, and W. A. Wood, J. Biol. Chem. 230, 457 (1958). 11R. P. Mortlock and W. A. Wood, J. Bacteriol. 88, 888 (1964). ~ W. J. Dobrogosz and R. D. DeMoss, J. Bacterlol. 85, 1350, 1356 (1963). M. Chakravorty, Biochim. Biophys. Acta 85, 152 (1964).
[106]
L--ARABINOSE ISOMERASE
599
Purification Procedures from Aerobacter aerogenes, PRL-R3
Growth Medium. The medium is composed of 0.2% (NH4)2S0~, 0.7% K2HP04, 0.3% KH2P04, 0.01~ MgSO~, 0.2% yeast extract, 0.2% peptone, 0.05% MnC12.4 H20, and 0.2% L-arabinose. MgSO~, MnC12, and L-arabinose solutions are sterilized separately in concentrated form and are added just before inoculation. Manganese ions form a precipitate with the phosphate buffer, but T.-arabinose isomerase is produced in greater amounts than in the same medium without manganese. Culture of Bacteria. The bacterium is inoculated into 5 ml of medium in a test tube and cultured for one night at 30 ° with shaking. One-tenth milliliter of culture is then transferred to 500 ml of the same medium in a Fernbach flask and grown for 20-24 hours on a rotary shaker. Twohundred and fifty milliliters of this culture is added to 20 liters of the medium in a glass carboy equipped with an aeration device, and the culture is incubated for 16 hours at 20 °. The cells are harvested in a Sharples centrifuge and washed with water. Preparation of Cell-Free Extract. The cells are suspended in 10-a M ethylenediaminetetraacetic acid (EDTA), pH 7.0, and disrupted in a 10-kc sonic oscillator for 10 minutes. The cell-free extract was obtained by centrifugation. Protamine Treatment. The extract is diluted to a protein concentration of 10 mg/ml by addition of 10-3 M EDTA, pH 7.0. Solid ammonium sulfate is added to 0.2 M in final concentration, and 0.2 volume of 2% protamine sulfate is added to precipitate the nucleic acids. The precipitate is removed by centrifugation (protamine supernatant). First Ammonium Sulfate Fractionation. Solid ammonium sulfate is added to give 45% saturation. The precipitate is removed by centrifugation, and additional ammonium sulfate is added to give 80% saturation. The precipitate was dissolved in 0.01 M Tris-acetate buffer, pH 7.0, and dialyzed overnight against the same buffer containing 10-3 M EDTA (ammonium sulfate I). Heat Treatment. MnCl~ solution is added to the above fraction to give 5 X 10-3 M concentration. The fraction in a flask is immersed in a water bath at 80 ° and stirred until the temperature of the solution reaches 49 °. It is then transferred to a bath at 50 ° for 10 minutes. After cooling, the precipitate is removed by centrifugation. Solid ammonium sulfate is added to the clear supernatant to obtain the precipitate between 45 and 75% saturation. The precipitate was collected, dissolved, and dialyzed against 0.01 M Tris-acetate buffer, pH 7.0, containing 5 X 10-3 M MnC12. NADH oxidase activity was almost completely removed in this step (heat fraction I).
600
[105]
ISOMERASES AND EPIMERASES
Acetone Fractionation. Acetone at --20 ° is slowly added while the temperature of the fraction is slowly lowered to --5 ° . Enzymatic activity is recovered mainly in the precipitate obtained between 20--30% and 30-40% acetone (acetone fraction). The precipitates were dissolved in 0.01 M Tris-acetate buffer, p H 7.0, and incubated at 55 ° for 5 minutes in the presence of 5 X 10-3 M of MnC12. The precipitate was removed by centrifugation (heat fraction I I ) . DEAE-Cellulose Chromatography. Enzyme was absorbed onto a column of DEAE-cellulose (3.8 X 25 em) which was equilibrated with 0.02 M Tris-acetate buffer, p H 7.0, and eluted by a linear gradient of 0 to 0.4 M of NaC1 in the same buffer. Enzyme was recovered at around 0.2 M of NaC1. The active fractions (nos. 72-85) were pooled, and solid ammonium sulfate was added to obtain the precipitates between 45 and 70% saturation. This preparation is almost free of L-ribulokinase activity (DEAE-cellulose fraction I). Sephadex G-IO0 Chromatography. The enzyme fraction is absorbed on a column of Sephadex G-100 (2.3 X 25 cm). The active fractions were collected by eluting 0.05 M NaC1 (Sephadex fraction). Second DEAE-Cellulose Chron~atography. Rechromatography on a column of DEAE-cellulose (1.4 X 22 cm) was carried out by the procedure described in the first DEAE-cellulose chromatography. The active fractions were combined and concentrated by the addition of solid ammonium sulfate (DEAE-cellulose fraction I I ) . The purification procedure is summarized in Table I. The final preparation represents an 83-fold enrichment of the specific activity and is essentially free from N A D H oxidase, ATPase, L-ribulokinase, D-ribulokinase, D-xylulokinase, ribitol dehydrogenase, L-ribulose 5-phosphate 4epimerase, D-ribulose 5-phosphate 3-epimerase, and phosphoketolase activities. TABLE I PURIFICATION OF L-ARABINOSE ISOMERASE FROM A. aerogenes (SFEcTROFHOTOMETRIC ASSAY)
Fraction
Protein
Crude extract Ammonium sulfate I Heat fraction I Ammonium sulfate II Acetone fraction Heat fraction II DEAE-cellulose fraction I Sephadex G-100 fraction DEAE-cellulose fraction II
56,200 20,350 10,300 8,440 3,900 1,890 720 460 153
Specific activity Units X 103 (units/mg of protein) 628 468 620 577 299 281 203 201 141
11 23 60 68 76 148 282 439 923
[105]
L-ARABINOSE ISOMERASE
601
Purification Procedure for Lactobacillus gayonii The procedure given is that described by Smyrniotis,2 but with some modifications. Growth Medium. A manganese-fortified medium yielded about 20 times more L-arabinose isomerase than the basal medium without manganese.TM The composition of the medium is as follows: 1% peptone, 0.2% yeast extract, 1% sodium acetate, 0.05% MnS04.4 H20, 0.02% MgSO~.7 H~O, 0.001% NaC1, 0.1% L-arabinose, and 0.5% glucose. Culture of Bacteria. See D-xylose isomerase. 7 For maximum production of the isomerase by L. gayonii, incubation is carried out for about 40 hours with glucose or 16 hours with L-arabinose at 32 ° for 20 liters of medium. Preparation Procedures. See Vol. V [42]. Further purification can be obtained by using heat treatment and by column chromatography on DEAE-cellulose procedures for A. aerogenes (above). The purification procedure is summarized in Table II. TABLE II PURIFICATION OF L-ARABINOSE ISOMERASE FROM L. gayonii (BASED UPON THE COLORIMETRIC ASSAY)
Fraction Crude extract Mn-treated fraction Ammonium sulate fraction (0.6-0.9 saturfation) Acetone fraction I)EAE-cellulose fraction
Protein (mg)
Units
Specific activity (micromoles/mg/10min)
255 162 24
1670 2115 1110
5.9 13.1 46.6
15 6
1024 770
85.5 140
Properties These properties are shown by the purified preparation from A.
aerogenes unless otherwise noted. Ef]ect of pH. The maximum activity was attained at pH 6.4--6.9 (A. aerogenes), 7.8--8.2 (L. gayonii) at 35 ° for 10 minutes of incubation. Stability. Enzyme is stable between pH 6.6 and 9.5 at 30 ° for 2 hours. Manganese ions protect against the thermal inactivation. Substrate Specificity and Affinity. Ketoses were produced from L-arabinose, n-galactose, or D-fucose, but not from other pentoses or from hexoses. The apparent Michaelis values from the Lineweaver-Burk plots 14K. Yamanaka and T. Higashihara, Agr. Biol. Chem. Japan 26, 162 (1962) (in English).
602
ISOMERASES AND EPIMERASES
[107]
are 3.3 X 10-2 M for L-arabinose, 0.27 M for D-fucose, and 0.37 M for n-galactose (pH 6.9, 38 °, 10 minutes). The ratio of relative activities toward these sugars (L-arabinose, D-fucose, and D-galactose) was 1:0.22: 0.23 (500 micromoles, of substrate, 50 °, 30 units of isomerase). These three sugars have the same configuration from C-1 to C-4. However, L-lyxose and L-ribose were not isomerized by this enzyme.15 Eight strains of heterofermentative lactic acid bacteria grown on L-arabinose had the same ratio of isomerase activities on L-arabinose and n-galactose.1¢ These activities were not separable by ammonium sulfate fractionation or by chromatography on DEAE-cellulose. The optimum pH for isomerization of L-arabinose, D-galactose, and D-fucose is identical at pH 6.4-6.9. From these observations, it may be concluded that this enzyme has one site with a high affinity for L-arabinose, but low affinity for D-galactose and D-fucose. The isomerase thus has the affinity for sugars with an L-c/s hydroxyl configuration at C-3 and C-4. Metal Requirement. The isomerase from both sources required Mn +÷ specifically for activity. Inhibitors. L-Arabitol and ribitol inhibit competitively the isomerization of all three aldoses. With L-arabinose as substrate, the K~ values for L-arabitol or ribitol are about 2.3 X 10-3 M and 3.5 X 10-4M, respectively. 15K. Yamanaka and W. A. Wood, unpublished observation, 1964. leK. Yamanaka, unpublished observation, 1962.
[ 107] 4 - D e o x y - L - t / n - e o - 5 - h e x o s u l o s e U r o n i c A c i d I s o m e r a s e 1 B y JACK PREISS
CHO(CH2OH)~CH~COCOOH~ CH~OHCOCHOHCH~COCOOH 4-Deoxy-L-threo-53-Deox~-D-glycero-2,5hexosuloseuronic hexodmlosonicacid acid (I)
(II)
Assay M e t h o d
Principle. The most convenient assay for the isomerization of compound (I) to compound (II) proved to be the spectrophotometric determination of the reduction of the latter by DPNH at 340 mg in the presence of an excess of 3-deoxy-n-glycero-2,5-hexodiulosonic acid reductase. The preparation of this enzyme, free from contamination with 1j. Preiss and G. Ashwell, J. Biol. Chem. 238, 1577 (1963).
ISOMERASES AND EPIMERASES
[106]
Properties ~
Substrate Specificity. D-Lyxose isomerase has been found only in cells grown on D-lyxose; D-mannose and D-fructose do not serve as inducers. However, the purified enzyme isomerizes D-mannose to D-fructose. The K,~ values for D-lyxose and D-mannose are 3.6 m M and 10 mM, respectively. The Vmax is 2.4 times greater with D-lyxose than with D-mannose. D-Ribose, D- or L-xylose, D- or L-arabinose, D-glucose, D-glucose 6-phosphate, and n-mannose 6-phosphate are not isomerized at a detection level of 0.5% the rate on D-lyxose. Activators and Inhibitors. Activity is nil in the absence of a metal ion. At 1 m M concentrations, the relative activity with various metal salts is as follows: MnC12, 100; FeClD, 78; FeSO~, 18; CaC12, 14; CoCl~, 8; MgC12, 0; ZnS04, 0; CuSO4, 0. The K,~ for Mn ÷÷ is 4 vM. Activity is nil in the presence of 10 ~ / p - c h l o r o m e r c u r i b e n z o a t e ; the activity cannot be restored with the further addition of reduced glutathione, and reduced glutathione was never found to be protective or stimulatory during fractionation or assay. Iodoacetate, oxidized glutathione, 2,6-dichlorophenolindophenol, or KDFe (CN)6 at. 1 m M and pH 7.0 showed no inhibition in the standard assay. Ef]ect of pH. The pH optimum is about 7.0. Activity as a function of pH varies with the buffer, being half maximal at pH 6.3 in maleate and at pH 4.9 in caeodylate. Molecular Weight. The sedimentation coefficient as determined by density gradient centrifugation is 3.7 to 3.8 S, suggesting a molecular weight of about 40,000.
[ 106] L - A r a b i n o s e I s o m e r a s e
By K. YAMANAKAand W. A. WOOD I,-Arabinose ~- L-rihulose This enzyme has been described by Cohen 1 briefly and by Smyrniotis 2 and by Horeekeff '~ in more detail. 1S. S. Cohen, see Vol. I [52]. 2p. Z. Symrniotis, see Vol. V [42]. 8B. L. Horecker, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), p. 178. Academic Press, New York, 1963. E. C. Heath, B. L. Horecker, P. Z. Smyrniotis, and Y. Takagi, J. Biol. Chem. 231, 1031 (1958).
[105]
L-ARABINOSE ISOMERASE
597
Assay Methods 2
Principle. L-Arabinose isomerase activity is assayed speetrophotometrically at 30 ° using a standard spectrophotometer equipped with an absorbancy converter, automatic cuvette positioner, and recorder. ~ The assay system is based on the following sequences of reactions:
L-Arabinose L-Ribulose -t- ATP
L-arabinose isomerase
~L-ribulose
L-ribulokinase
, L-ribulose5-phosphate -{- ADP
Phosphoenolpyruvate ~ ADP Pyruvate ~ NADH
pyruvate kinase
) pyruvate T ATP
lactate dehydrogenase
, lactate ~- NAD
Reagents
Maleate buffer, 0.4 M, pH 6.9 MgC12, 0.1 M ATP, 0.1 M, adjusted to pH 6.9 Phosphoenolpyruvate, 0.025 M, adjusted to pH 6.9 Sodium glutathione NADH, 0.01 M L-Arabinose, 0.1 M Purified L-ribulokinase, free from L-arabinose isomerase6 Lactate dehydrogenase-pyruvate kinase, crystalline (Worthington) All solutions are prepared separately and kept in a deepfreeze. A reagent stock solution is then prepared by mixing 0.8 ml of maleate buffer, 0.4 ml of MgC12, 0.2 ml of ATP, 0.4 ml of phosphoenolpyruvate, 18 mg of glutathione, 0.4 ml of NADH, 0.05 ml of lactate dehydrogenase, and water to a total volume of 4.0 ml. This mixture can be kept frozen and prepared fresh every 3 days. Procedures. To a microcuvette of 0.5-ml capacity and 1-em light path are added 0.1 ml of reagent mixture, 2 units of purified L-ribulokinase, 2 mieromoles of L-arabinose, suitable amounts of L-arabinose isomerase preparation, and water to a volume of 0.15 ml. Blanks are run for NADH oxidase with buffer and NADH, and for ATPase activity by omitting L-ribulokinase and L-arabinose. L-Ribulokinase activity may be determined by the same method by adding L-ribulose instead of L-arabinose. NADH oxidase and ATPase activities are subtracted from the total activity. A linear relationship between the rate of NADH ~W. A. Wood and S. R. Gilford, Anal. Biochem. 2, 589, 601 (1961). 6See this volume [80]. See also F. J. Simpson and W. A. Wood, J. Biol. Chem. 23~), 473 (1958).
598
ISOMERASES AND EPIMERASES
[105]
oxidation and amount of enzyme is obtained between 0 and 0.3 optical density units per minute. This corresponds to 0-0.067 unit of isomerase. Alternate Assay. For crude preparations, the isomerase activity cannot be determined by a spectrophotometric method owing to the strong interference of NADH oxidase and ATPase. In that case, the following procedure is followed: The reaction mixture (1.0 ml) contains 0.5 ml of 0.05M maleate buffer, pH 6.9, for Aerobacter enzyme or of 0.05 M Tris-HCl buffer, pH 8.0, for Lactobacillus enzyme, 0.05 ml of 0.1 M MnCl~ (or MnSO,), and 0.01--0.1 ml of enzyme preparation with adequate dilution. After incubation at 35 ° for 5 minutes, the reaction is started by adding 0.05 ml of 0.1 M L-arabinose, and incubation is continued at 35 ° for 10 minutes. L-Ribulose is determined by the cysteine-carbazole procedures using T,-ribulose-o-nitrophenylhydrazone as a standard. Definition of Unit and Specific Activity. A unit of enzyme is defined as the amount required to effect an absorbancy change of 1.0 per minute at 340 m~ under these conditions. Specific activity is expressed as units per milligram of protein. For the colorimetrie procedure, see Vol. V [42] and the procedure for D-xylose isomerase2 Sources L-Arabinose isomerase activity has been found in the cells grown on T.-arabinose for the following organisms: Lactobacillus plantarum, ",~ some strains of homolaetie acid bacteria related to L. plantarum, 9 and of Aerobacter aerogenes. 1°,11 This enzyme is also produced in L. plantarum grown on D-xylose. ~ However, many of the heterofermentative lactic acid bacteria, such as L. gayonii, L. mannitopous, L. buchneri, L. brevis, L. pen~oaceticus, and L. lycopersici, and some of the Pediococcus group also produce L-arabinose isomerase from the media containing glucose, fructose, galactose, n-xylose, L-arabinose, or malt extract. 9 The glucosegrown cells of L. gayonii contain T,-arabinose isomerase but no L-ribulokinase and NADH oxidase. Induction of L-arabinose isomerase by Pediococcus pentosaceus ~ and by Lactobacillus plantarum ~ was reported. TK. Yamanaka, see this volume [104]. s j. O. Lampen, Proc. Am. Chem. Soe., 44c (1954). 9K. Yamanaka, Bull. Agr. Chem. Soc. Japan 24, 310, (1960) (in English); Mere. Fac. Agr., Kagawa Univ., No. 16 (1963) (in English). •F. J. Simpson, M. J. Wolin, and W. A. Wood, J. Biol. Chem. 230, 457 (1958). 11R. P. Mortlock and W. A. Wood, J. Bacteriol. 88, 888 (1964). ~ W. J. Dobrogosz and R. D. DeMoss, J. Bacterlol. 85, 1350, 1356 (1963). M. Chakravorty, Biochim. Biophys. Acta 85, 152 (1964).
[106]
L--ARABINOSE ISOMERASE
599
Purification Procedures from Aerobacter aerogenes, PRL-R3
Growth Medium. The medium is composed of 0.2% (NH4)2S0~, 0.7% K2HP04, 0.3% KH2P04, 0.01~ MgSO~, 0.2% yeast extract, 0.2% peptone, 0.05% MnC12.4 H20, and 0.2% L-arabinose. MgSO~, MnC12, and L-arabinose solutions are sterilized separately in concentrated form and are added just before inoculation. Manganese ions form a precipitate with the phosphate buffer, but T.-arabinose isomerase is produced in greater amounts than in the same medium without manganese. Culture of Bacteria. The bacterium is inoculated into 5 ml of medium in a test tube and cultured for one night at 30 ° with shaking. One-tenth milliliter of culture is then transferred to 500 ml of the same medium in a Fernbach flask and grown for 20-24 hours on a rotary shaker. Twohundred and fifty milliliters of this culture is added to 20 liters of the medium in a glass carboy equipped with an aeration device, and the culture is incubated for 16 hours at 20 °. The cells are harvested in a Sharples centrifuge and washed with water. Preparation of Cell-Free Extract. The cells are suspended in 10-a M ethylenediaminetetraacetic acid (EDTA), pH 7.0, and disrupted in a 10-kc sonic oscillator for 10 minutes. The cell-free extract was obtained by centrifugation. Protamine Treatment. The extract is diluted to a protein concentration of 10 mg/ml by addition of 10-3 M EDTA, pH 7.0. Solid ammonium sulfate is added to 0.2 M in final concentration, and 0.2 volume of 2% protamine sulfate is added to precipitate the nucleic acids. The precipitate is removed by centrifugation (protamine supernatant). First Ammonium Sulfate Fractionation. Solid ammonium sulfate is added to give 45% saturation. The precipitate is removed by centrifugation, and additional ammonium sulfate is added to give 80% saturation. The precipitate was dissolved in 0.01 M Tris-acetate buffer, pH 7.0, and dialyzed overnight against the same buffer containing 10-3 M EDTA (ammonium sulfate I). Heat Treatment. MnCl~ solution is added to the above fraction to give 5 X 10-3 M concentration. The fraction in a flask is immersed in a water bath at 80 ° and stirred until the temperature of the solution reaches 49 °. It is then transferred to a bath at 50 ° for 10 minutes. After cooling, the precipitate is removed by centrifugation. Solid ammonium sulfate is added to the clear supernatant to obtain the precipitate between 45 and 75% saturation. The precipitate was collected, dissolved, and dialyzed against 0.01 M Tris-acetate buffer, pH 7.0, containing 5 X 10-3 M MnC12. NADH oxidase activity was almost completely removed in this step (heat fraction I).
600
[105]
ISOMERASES AND EPIMERASES
Acetone Fractionation. Acetone at --20 ° is slowly added while the temperature of the fraction is slowly lowered to --5 ° . Enzymatic activity is recovered mainly in the precipitate obtained between 20--30% and 30-40% acetone (acetone fraction). The precipitates were dissolved in 0.01 M Tris-acetate buffer, p H 7.0, and incubated at 55 ° for 5 minutes in the presence of 5 X 10-3 M of MnC12. The precipitate was removed by centrifugation (heat fraction I I ) . DEAE-Cellulose Chromatography. Enzyme was absorbed onto a column of DEAE-cellulose (3.8 X 25 em) which was equilibrated with 0.02 M Tris-acetate buffer, p H 7.0, and eluted by a linear gradient of 0 to 0.4 M of NaC1 in the same buffer. Enzyme was recovered at around 0.2 M of NaC1. The active fractions (nos. 72-85) were pooled, and solid ammonium sulfate was added to obtain the precipitates between 45 and 70% saturation. This preparation is almost free of L-ribulokinase activity (DEAE-cellulose fraction I). Sephadex G-IO0 Chromatography. The enzyme fraction is absorbed on a column of Sephadex G-100 (2.3 X 25 cm). The active fractions were collected by eluting 0.05 M NaC1 (Sephadex fraction). Second DEAE-Cellulose Chron~atography. Rechromatography on a column of DEAE-cellulose (1.4 X 22 cm) was carried out by the procedure described in the first DEAE-cellulose chromatography. The active fractions were combined and concentrated by the addition of solid ammonium sulfate (DEAE-cellulose fraction I I ) . The purification procedure is summarized in Table I. The final preparation represents an 83-fold enrichment of the specific activity and is essentially free from N A D H oxidase, ATPase, L-ribulokinase, D-ribulokinase, D-xylulokinase, ribitol dehydrogenase, L-ribulose 5-phosphate 4epimerase, D-ribulose 5-phosphate 3-epimerase, and phosphoketolase activities. TABLE I PURIFICATION OF L-ARABINOSE ISOMERASE FROM A. aerogenes (SFEcTROFHOTOMETRIC ASSAY)
Fraction
Protein
Crude extract Ammonium sulfate I Heat fraction I Ammonium sulfate II Acetone fraction Heat fraction II DEAE-cellulose fraction I Sephadex G-100 fraction DEAE-cellulose fraction II
56,200 20,350 10,300 8,440 3,900 1,890 720 460 153
Specific activity Units X 103 (units/mg of protein) 628 468 620 577 299 281 203 201 141
11 23 60 68 76 148 282 439 923
[105]
L-ARABINOSE ISOMERASE
601
Purification Procedure for Lactobacillus gayonii The procedure given is that described by Smyrniotis,2 but with some modifications. Growth Medium. A manganese-fortified medium yielded about 20 times more L-arabinose isomerase than the basal medium without manganese.TM The composition of the medium is as follows: 1% peptone, 0.2% yeast extract, 1% sodium acetate, 0.05% MnS04.4 H20, 0.02% MgSO~.7 H~O, 0.001% NaC1, 0.1% L-arabinose, and 0.5% glucose. Culture of Bacteria. See D-xylose isomerase. 7 For maximum production of the isomerase by L. gayonii, incubation is carried out for about 40 hours with glucose or 16 hours with L-arabinose at 32 ° for 20 liters of medium. Preparation Procedures. See Vol. V [42]. Further purification can be obtained by using heat treatment and by column chromatography on DEAE-cellulose procedures for A. aerogenes (above). The purification procedure is summarized in Table II. TABLE II PURIFICATION OF L-ARABINOSE ISOMERASE FROM L. gayonii (BASED UPON THE COLORIMETRIC ASSAY)
Fraction Crude extract Mn-treated fraction Ammonium sulate fraction (0.6-0.9 saturfation) Acetone fraction I)EAE-cellulose fraction
Protein (mg)
Units
Specific activity (micromoles/mg/10min)
255 162 24
1670 2115 1110
5.9 13.1 46.6
15 6
1024 770
85.5 140
Properties These properties are shown by the purified preparation from A.
aerogenes unless otherwise noted. Ef]ect of pH. The maximum activity was attained at pH 6.4--6.9 (A. aerogenes), 7.8--8.2 (L. gayonii) at 35 ° for 10 minutes of incubation. Stability. Enzyme is stable between pH 6.6 and 9.5 at 30 ° for 2 hours. Manganese ions protect against the thermal inactivation. Substrate Specificity and Affinity. Ketoses were produced from L-arabinose, n-galactose, or D-fucose, but not from other pentoses or from hexoses. The apparent Michaelis values from the Lineweaver-Burk plots 14K. Yamanaka and T. Higashihara, Agr. Biol. Chem. Japan 26, 162 (1962) (in English).
602
ISOMERASES AND EPIMERASES
[107]
are 3.3 X 10-2 M for L-arabinose, 0.27 M for D-fucose, and 0.37 M for n-galactose (pH 6.9, 38 °, 10 minutes). The ratio of relative activities toward these sugars (L-arabinose, D-fucose, and D-galactose) was 1:0.22: 0.23 (500 micromoles, of substrate, 50 °, 30 units of isomerase). These three sugars have the same configuration from C-1 to C-4. However, L-lyxose and L-ribose were not isomerized by this enzyme.15 Eight strains of heterofermentative lactic acid bacteria grown on L-arabinose had the same ratio of isomerase activities on L-arabinose and n-galactose.1¢ These activities were not separable by ammonium sulfate fractionation or by chromatography on DEAE-cellulose. The optimum pH for isomerization of L-arabinose, D-galactose, and D-fucose is identical at pH 6.4-6.9. From these observations, it may be concluded that this enzyme has one site with a high affinity for L-arabinose, but low affinity for D-galactose and D-fucose. The isomerase thus has the affinity for sugars with an L-c/s hydroxyl configuration at C-3 and C-4. Metal Requirement. The isomerase from both sources required Mn +÷ specifically for activity. Inhibitors. L-Arabitol and ribitol inhibit competitively the isomerization of all three aldoses. With L-arabinose as substrate, the K~ values for L-arabitol or ribitol are about 2.3 X 10-3 M and 3.5 X 10-4M, respectively. 15K. Yamanaka and W. A. Wood, unpublished observation, 1964. leK. Yamanaka, unpublished observation, 1962.
[ 107] 4 - D e o x y - L - t / n - e o - 5 - h e x o s u l o s e U r o n i c A c i d I s o m e r a s e 1 B y JACK PREISS
CHO(CH2OH)~CH~COCOOH~ CH~OHCOCHOHCH~COCOOH 4-Deoxy-L-threo-53-Deox~-D-glycero-2,5hexosuloseuronic hexodmlosonicacid acid (I)
(II)
Assay M e t h o d
Principle. The most convenient assay for the isomerization of compound (I) to compound (II) proved to be the spectrophotometric determination of the reduction of the latter by DPNH at 340 mg in the presence of an excess of 3-deoxy-n-glycero-2,5-hexodiulosonic acid reductase. The preparation of this enzyme, free from contamination with 1j. Preiss and G. Ashwell, J. Biol. Chem. 238, 1577 (1963).