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[97] l -arabinose isomerase from lactobacillus gayonii
458
[97]
EPIMERASES AND ISOMERASES
a lower molecular activity, enzyme synthesized without added Mn '-'÷ ion is more temperature labile than enzyme synthesized in the presence of added Mn 2÷ ion. Several divalent cations can confer temperature stability, but not full activity, to an enzyme synthesized without added Mn ~-÷. In contrast, of the ions tested, only Mn 2÷ is able to cause the synthesis of a fully active enzyme. Mn 2÷ is, however, unable to convert partially active enzyme to fully active enzyme either in vitro or in vivo.
[97] L - A r a b i n o s e I s o m e r a s e f r o m L a c t o b a c i l l u s
By
gayonH
KEI YAMANAKA
L-Arabinose .~- L-ribulose This enzyme from Lactobacillus gayonii has been described briefly) Comprehensive data on crystalline enzyme will be discussed in this section. Assay M e t h o d
L-Arabinose isomerase activity can be assayed either by the spectrephotometric method or by the cysteine-carbazole test as described in a previous paper2 Purification Procedure s
Culture. The growth medium and culture of the organism are the same as described previously. 1 The composition of medium is as follows: 1% peptone, 1% sodium acetate, 0.2% yeast extract, 0.02% MgS04.7H20, 0.01% MnSO~.4H20, 0.01% CoCI~'6H20, 1% u-glucose, and 0.1% L-arabinose. Preparation of Cell-Free Extracts. The washed L-arabinose-grown cells, 60 g in wet weight from 40 liters of medium, are disrupted in small portion by grinding with about 150-200 g of levigated alumina, 3 and the enzyme is extracted with about 650 ml of 20 m M Tris-HC1 buffer at p H 7.5. Alumina and cell debris are removed by centrifugation (crude extract, 594 ml). To the crude extract, 30 ml of 1 M MnC12 is added dropwise and pH is maintained at 7.0-7.5 by adjusting with 1 N NaOH. 1K. Yamanaka and W. A. Wood, see this series, Vol. 9 [106]. ' T. Nakamatu and K. Yamanaka, Biochim. Biophys. Acta 178, 156 (1969). 3Levigated alumina, about 300 mesh for chromatography was purchased from Wake Pure Chemicals, Osaka, Japan.
[97]
L-ARABINOSE ISOMERASE
459
After standing for 30 min, the precipitate is centrifuged and discarded (MnCl2-treated fraction, 610 ml). Ammonium SulIate Fractionation. The amount of ammonium sulfate is calculated from the table given by Green and Hughes 4 with a temperature correction of 0.92 for conversion to 0 °. To the manganese-treated fraction, 176 g of ammonium sulfate is added (50% saturation). The precipitate is discarded. The supernatant is treated with 195 g of ammonium sulfate (95% saturation). The precipitate is dissolved in 20 mM Tris.HC1 buffer (pH 7.5) and dialyzed overnight against the same buffer containing 5 mM MnSO, (102 ml). Heat Treatment. The enzyme solution in the presence of 5 mM MnC12 is immersed in a water bath at 80 °. When the temperature of the solution reaches 47 °, the flask is transferred to another water bath at 50 ° and maintained for 5 min at the same temperature. The fraction is cooled in an ice bath and the coagulated proteins are removed by centrifugation and discarded (98 ml). To the supernatant is added 70 g of ammonium sulfate to 100% saturation. The precipitate is dissolved in 20 mM Tris.HC1 buffer (pH 7.5) and dialyzed overnight with the same procedure as that described above (80 ml). Column Chromatography on DEAE-Cellulose. The enzyme is chromatographed on DEAE-cellulose (3.0 cm )K 70 cm) which had been equilibrated with 20 mM Tris.HC1 buffer at pH 7.5. The proteins are eluted with a linear gradient of KC1 between 0 and 0.6 M at pH 7.5. Active fractions are pooled and the enzyme precipitated with ammonium sulfate to 90% of saturation. The enzyme is dissolved, passed through a column of Sephadex G-200 (1.5 cm X 90 cm) and eluted with 20 mM Tris.HC1 buffer at pH 7.5. Active fractions are combined (17.6 ml), and the enzyme is precipitated with ammonium sulfate to 80% saturation. Crystallization. Crystallization of the enzyme is carried out with the addition of saturated ammonium sulfate solution. After overnight dialysis, 0.98 ml of a saturated ammonillm sulfate is added to the enzyme solution (1.15 ml, protein concentration is about 3.5%). Ammonium sulfate content reached is 46% saturation. The precipitate is removed by centrifugation. The ammonium sulfate content is slowly increased at a rate of not more than 0.05 saturation per day until 56% saturation is reached. A trace of precipitate is removed at each step by centrifugation. The amorphous precipitate between 0.51 and 0.558 saturation contains a small amount of the L-arabinose isomerase activity. Finally, the solution is brought to 63% saturation and the clear solution held at 5 ° for 4 days. The crystals are collected by centrifugation and dissolved in 2.5 4A. A. Green and W. L. Hughes, see this series, Vol. 1 [10].
460
[97]
EPIMERASES AND ISOMERASES
TABLE I PURIFICATION OF L-ARABINOSE ISOMERASE
Fraction Crude extract MnCl2-treated fraction Ammonium sulfate fraction (50-95% saturation) Mn-heated fraction DEAE-cellulose eluate Sephadex G-200 eluate Crystals
Total protein (mg)
Total units"
Specific activity (t~moles/mg/min)
Yield (%)
2380 1340 1400
1188 1390 1420
0.50 1.03 1.01
117 119
1360 158 67 45
1190 530 364 239
0.88 3.32 5.44 5.31
100 45 33 20
" Colorimetric units. ml of 20 m M Tris.HC1 buffer at p H 7.5 and dialyzed against 100 ml of the same buffer containing 5 m M MnSO4 overnight (first crystals). Recrystallization is performed with the same procedure at 63% saturation. The purification procedure is summarized in Table I.
Properties 2
Purity. The second crystals show a single symmetrical moving peak on ultracentrifugation. Molecular Weight. The molecular weight is estimated by centrifugation in a sucrose density gradient by the method of M a r t i n and Ames 5 as 2 . 7 1 X 10 ~. Ej~ect o] pH. The m a x i m u m activity is attained at p H 6.0-7.0 at 35 ° for 10 min of incubation. Stability. The enzyme is stable at p H 5.5-9.0 for l0 rain of incubation at 50 ° . Substrate Specificity. The crystalline enzyme is specific for L-arabinose and L-ribulose. Other pentoses and hexoses are inactive for the enzyme. Metal Requirement. The enzyme requires Mn '-,+ for activity. The solution of second crystals is dialyzed against 10 m M T r i s . HC1 buffer at p H 8.0 containing 5 m M E D T A at 2 ° for 48 hr, then dialyzed against the same buffer without E D T A for 24 hr. The activity of the dialyzed enzyme preparation can be recovered specifically by the addition of M n 2÷, and cobaltous ion activates to about half the extent of M n ~÷. Potassium 5 R. G. Martin and B. N. Ames, J. Biol. Chem. 236, 1372 (1961).
[97]
L-ARABINOSE ISOMERASE
461
TABLE II INHIBITION CONSTANTS OF PENTITOLS FOR L-ARABINOSE ISOMERASE
Inhibition constant (Ki)(raM) Sources
Ribitol
Lactobacillus gayonii a Aerobacter aerogenes, PRL-R3 b A. aerogenes, M-7 ~ Escherichia colie Streptomyces/ Clostridium acetobutylicumg
6 0.35 1.6 6 1.0 ND
L-Arabitol Xylitol 7.5 2.3 2.2 18 1.1 23
38 ND c 27 ND 15 ND
T. Nakamatu and K. Yamanaka, Biochim. Biophys. Acta 178, 156 (1969). b K. Yamanaka and W. A. Wood, see this series, Vol. 9 [106]. c ND, not determined. d K. Izumori and K. Yamanaka, J. Ferment. Technol. 51, 452 (1973). e j. W. Patrick and N. Lee, J. Biol. Chem. 234, 4312 (1968). i K. Yamanaka and K. Izumori, Agr. Biol. Chem. 37, 521 (1973). M. Tomoeda, H. Horitsu, and I. Sasaki, Agr. Biol. Chem. 33, 151 (1969). and strontium ions have a slight activation effect, but N a +, Li +, M g 2+, and Ba -~+ are ineffective. Zn 2+, Cu 2+, Fe 2+, and H g 2+ are inhibitory. The Michaelis constant for Mn -~+is 5.25 tLM. E f f e c t o/ S u b s t r a t e Concentration. The reaction mechanism of L-arabinose isomerase is compulsory ordered and involves the formation of a t e r n a r y complex of enzyme-Mn-substrate. Since dialysis against E D T A appears to yield a Mn-free enzyme preparation, the apparent Michaelis constant for L-arabinose is determined with 5, 10, 20, and 100 t~M of Mn 2+. These calculated apparent Michaelis constants are then plotted vs the reciprocal of the concentration of Mn 2+. From the intercept of this line, the true Michaelis constant for L-arabinose is obtained as 55 mM. The affinity for L-ribulose is calculated as 5.0 m M from the conventional Lineweaver-Burk equation. I n h i 6 i t i o n b y Pentitols. Activity of the crystalline L-arabinose isomerase is competitively inhibited by structurally related pentitols. The Ki values of pentitols for the isomerase from several bacterial sources are summarized in T a b l e II. The enzyme is most sensitive to ribitol for all species, and L-arabitol is the most potent inhibitor. D-Arabitol does not inhibit the enzyme activity for all species.
[97]
EPIMERASES AND ISOMERASES
a lower molecular activity, enzyme synthesized without added Mn '-'÷ ion is more temperature labile than enzyme synthesized in the presence of added Mn 2÷ ion. Several divalent cations can confer temperature stability, but not full activity, to an enzyme synthesized without added Mn ~-÷. In contrast, of the ions tested, only Mn 2÷ is able to cause the synthesis of a fully active enzyme. Mn 2÷ is, however, unable to convert partially active enzyme to fully active enzyme either in vitro or in vivo.
[97] L - A r a b i n o s e I s o m e r a s e f r o m L a c t o b a c i l l u s
By
gayonH
KEI YAMANAKA
L-Arabinose .~- L-ribulose This enzyme from Lactobacillus gayonii has been described briefly) Comprehensive data on crystalline enzyme will be discussed in this section. Assay M e t h o d
L-Arabinose isomerase activity can be assayed either by the spectrephotometric method or by the cysteine-carbazole test as described in a previous paper2 Purification Procedure s
Culture. The growth medium and culture of the organism are the same as described previously. 1 The composition of medium is as follows: 1% peptone, 1% sodium acetate, 0.2% yeast extract, 0.02% MgS04.7H20, 0.01% MnSO~.4H20, 0.01% CoCI~'6H20, 1% u-glucose, and 0.1% L-arabinose. Preparation of Cell-Free Extracts. The washed L-arabinose-grown cells, 60 g in wet weight from 40 liters of medium, are disrupted in small portion by grinding with about 150-200 g of levigated alumina, 3 and the enzyme is extracted with about 650 ml of 20 m M Tris-HC1 buffer at p H 7.5. Alumina and cell debris are removed by centrifugation (crude extract, 594 ml). To the crude extract, 30 ml of 1 M MnC12 is added dropwise and pH is maintained at 7.0-7.5 by adjusting with 1 N NaOH. 1K. Yamanaka and W. A. Wood, see this series, Vol. 9 [106]. ' T. Nakamatu and K. Yamanaka, Biochim. Biophys. Acta 178, 156 (1969). 3Levigated alumina, about 300 mesh for chromatography was purchased from Wake Pure Chemicals, Osaka, Japan.
[97]
L-ARABINOSE ISOMERASE
459
After standing for 30 min, the precipitate is centrifuged and discarded (MnCl2-treated fraction, 610 ml). Ammonium SulIate Fractionation. The amount of ammonium sulfate is calculated from the table given by Green and Hughes 4 with a temperature correction of 0.92 for conversion to 0 °. To the manganese-treated fraction, 176 g of ammonium sulfate is added (50% saturation). The precipitate is discarded. The supernatant is treated with 195 g of ammonium sulfate (95% saturation). The precipitate is dissolved in 20 mM Tris.HC1 buffer (pH 7.5) and dialyzed overnight against the same buffer containing 5 mM MnSO, (102 ml). Heat Treatment. The enzyme solution in the presence of 5 mM MnC12 is immersed in a water bath at 80 °. When the temperature of the solution reaches 47 °, the flask is transferred to another water bath at 50 ° and maintained for 5 min at the same temperature. The fraction is cooled in an ice bath and the coagulated proteins are removed by centrifugation and discarded (98 ml). To the supernatant is added 70 g of ammonium sulfate to 100% saturation. The precipitate is dissolved in 20 mM Tris.HC1 buffer (pH 7.5) and dialyzed overnight with the same procedure as that described above (80 ml). Column Chromatography on DEAE-Cellulose. The enzyme is chromatographed on DEAE-cellulose (3.0 cm )K 70 cm) which had been equilibrated with 20 mM Tris.HC1 buffer at pH 7.5. The proteins are eluted with a linear gradient of KC1 between 0 and 0.6 M at pH 7.5. Active fractions are pooled and the enzyme precipitated with ammonium sulfate to 90% of saturation. The enzyme is dissolved, passed through a column of Sephadex G-200 (1.5 cm X 90 cm) and eluted with 20 mM Tris.HC1 buffer at pH 7.5. Active fractions are combined (17.6 ml), and the enzyme is precipitated with ammonium sulfate to 80% saturation. Crystallization. Crystallization of the enzyme is carried out with the addition of saturated ammonium sulfate solution. After overnight dialysis, 0.98 ml of a saturated ammonillm sulfate is added to the enzyme solution (1.15 ml, protein concentration is about 3.5%). Ammonium sulfate content reached is 46% saturation. The precipitate is removed by centrifugation. The ammonium sulfate content is slowly increased at a rate of not more than 0.05 saturation per day until 56% saturation is reached. A trace of precipitate is removed at each step by centrifugation. The amorphous precipitate between 0.51 and 0.558 saturation contains a small amount of the L-arabinose isomerase activity. Finally, the solution is brought to 63% saturation and the clear solution held at 5 ° for 4 days. The crystals are collected by centrifugation and dissolved in 2.5 4A. A. Green and W. L. Hughes, see this series, Vol. 1 [10].
460
[97]
EPIMERASES AND ISOMERASES
TABLE I PURIFICATION OF L-ARABINOSE ISOMERASE
Fraction Crude extract MnCl2-treated fraction Ammonium sulfate fraction (50-95% saturation) Mn-heated fraction DEAE-cellulose eluate Sephadex G-200 eluate Crystals
Total protein (mg)
Total units"
Specific activity (t~moles/mg/min)
Yield (%)
2380 1340 1400
1188 1390 1420
0.50 1.03 1.01
117 119
1360 158 67 45
1190 530 364 239
0.88 3.32 5.44 5.31
100 45 33 20
" Colorimetric units. ml of 20 m M Tris.HC1 buffer at p H 7.5 and dialyzed against 100 ml of the same buffer containing 5 m M MnSO4 overnight (first crystals). Recrystallization is performed with the same procedure at 63% saturation. The purification procedure is summarized in Table I.
Properties 2
Purity. The second crystals show a single symmetrical moving peak on ultracentrifugation. Molecular Weight. The molecular weight is estimated by centrifugation in a sucrose density gradient by the method of M a r t i n and Ames 5 as 2 . 7 1 X 10 ~. Ej~ect o] pH. The m a x i m u m activity is attained at p H 6.0-7.0 at 35 ° for 10 min of incubation. Stability. The enzyme is stable at p H 5.5-9.0 for l0 rain of incubation at 50 ° . Substrate Specificity. The crystalline enzyme is specific for L-arabinose and L-ribulose. Other pentoses and hexoses are inactive for the enzyme. Metal Requirement. The enzyme requires Mn '-,+ for activity. The solution of second crystals is dialyzed against 10 m M T r i s . HC1 buffer at p H 8.0 containing 5 m M E D T A at 2 ° for 48 hr, then dialyzed against the same buffer without E D T A for 24 hr. The activity of the dialyzed enzyme preparation can be recovered specifically by the addition of M n 2÷, and cobaltous ion activates to about half the extent of M n ~÷. Potassium 5 R. G. Martin and B. N. Ames, J. Biol. Chem. 236, 1372 (1961).
[97]
L-ARABINOSE ISOMERASE
461
TABLE II INHIBITION CONSTANTS OF PENTITOLS FOR L-ARABINOSE ISOMERASE
Inhibition constant (Ki)(raM) Sources
Ribitol
Lactobacillus gayonii a Aerobacter aerogenes, PRL-R3 b A. aerogenes, M-7 ~ Escherichia colie Streptomyces/ Clostridium acetobutylicumg
6 0.35 1.6 6 1.0 ND
L-Arabitol Xylitol 7.5 2.3 2.2 18 1.1 23
38 ND c 27 ND 15 ND
T. Nakamatu and K. Yamanaka, Biochim. Biophys. Acta 178, 156 (1969). b K. Yamanaka and W. A. Wood, see this series, Vol. 9 [106]. c ND, not determined. d K. Izumori and K. Yamanaka, J. Ferment. Technol. 51, 452 (1973). e j. W. Patrick and N. Lee, J. Biol. Chem. 234, 4312 (1968). i K. Yamanaka and K. Izumori, Agr. Biol. Chem. 37, 521 (1973). M. Tomoeda, H. Horitsu, and I. Sasaki, Agr. Biol. Chem. 33, 151 (1969). and strontium ions have a slight activation effect, but N a +, Li +, M g 2+, and Ba -~+ are ineffective. Zn 2+, Cu 2+, Fe 2+, and H g 2+ are inhibitory. The Michaelis constant for Mn -~+is 5.25 tLM. E f f e c t o/ S u b s t r a t e Concentration. The reaction mechanism of L-arabinose isomerase is compulsory ordered and involves the formation of a t e r n a r y complex of enzyme-Mn-substrate. Since dialysis against E D T A appears to yield a Mn-free enzyme preparation, the apparent Michaelis constant for L-arabinose is determined with 5, 10, 20, and 100 t~M of Mn 2+. These calculated apparent Michaelis constants are then plotted vs the reciprocal of the concentration of Mn 2+. From the intercept of this line, the true Michaelis constant for L-arabinose is obtained as 55 mM. The affinity for L-ribulose is calculated as 5.0 m M from the conventional Lineweaver-Burk equation. I n h i 6 i t i o n b y Pentitols. Activity of the crystalline L-arabinose isomerase is competitively inhibited by structurally related pentitols. The Ki values of pentitols for the isomerase from several bacterial sources are summarized in T a b l e II. The enzyme is most sensitive to ribitol for all species, and L-arabitol is the most potent inhibitor. D-Arabitol does not inhibit the enzyme activity for all species.