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[36] Ribitol 5-phosphate dehydrogenase
240
ENZYMES OF SUGAR ACTIVATION
[36]
amplitude is a saturating function of the substrate concentration. The half-band width is the same as that for free DPNH. These data are consistent with the hypothesis that the epimerization reaction involves oxidation of the hexose residue at carbon 4 by the bound DPN to form a ketone that becomes symmetrically placed relative to the resulting D P N H so that subsequent reduction of the ketone by that D P N H can yield either a galactose or a glucose residue. The effective pH range for the enzyme is large. At pH 6.35, 7.15, 8.00, 8.50, and 9.64, the relative activities at 27 ° were 0.88, 0.90, 1.00, 1.00, and 0.79, respectively. The Km and the turnover number (k3) for UDPgalaetose under the standard assay conditions equal 1.6 X 10-4M and 5.0 X 102 sec -1, respectively. Imae, Morikawa, and Kurahashi, is using a partially purified preparation of E. coli UDP-galactose 4-epimerase, measured a K~ for UDP-glucose of 1.0 X 10-3 M at 25 ° and pH = 8.7. The values found by these workers for the equilibrium constant, the pH optimum and the K~ for UDP-galactose are in good agreement with those given above. The activation energy for catalysis of the UDPgalactose to UDP-glucose reaction in the temperature range 10-25 ° is 10.5 kcal/mole; this results in a 1.85-fold increase in rate per 10° increase in temperature. 18y. Imae, N. Morikawa, and K. Kurahashi, J. Biochem. (Tokyo) 56, 138 (1964).
[36]
Ribitol 5-Phosphate Dehydrogenase 1
By D-Ribitol 5-phosphate +
L u i s GLASER
TPN + TPNH or ~ Doribulose5-phosphate -t- or -t- H+ DPN + DPNH
Assay Method
Principle. The reaction is measured by determining the absorbanee change at 340 m~ due to the formation of TPNH in the forward direction or the disappearance of T P N H in the reverse reaction. Reagents D-Ribulose 5-P, 0.003 M, prepared by the method of I-Iorecker 2 'L. Glaser, Biochim. Biophys. Acta 67, 525 (1963). :B. L. Horecker, Vol. III [29].
[35]
RIBITOL 5-PHOSPHATE DEHYDROGENASE
241
D-Ribitel-5-P, O.02M, prepared from D-ribose-5-P by reduction with NaBH43, 4 T P N H , 0.001 M, prepared daily in 0.05 M Tris-C1, pH 8.0 T P N , 0.05 M, p H 7.0 Ammonium acetate, 1 M, adjusted to pH 7.8 by the addition of NH40H 0.05 M Tris-C1--0.001 M E D T A , p H 7.8
Procedure AssAY A. REDUCTION OF D-RIBULOSE-5-P. The reaction mixtures contain 0.2 ml of T r i s - C I - E D T A buffer, 0.1 ml of ribulose-5-P, 0.1 ml of T P N H , 0.5 ml of ammonium acetate and enzyme in a final volume of 1 ml. Control reaction mixtures contain all the components minus Dribulose-5-P. The decrease in absorbancy at 340 m~ is measured at 25 ° after addition of the enzyme. In all but the purest fractions D-ribose-5-P can replace D-ribulose-5-P since the enzyme is contaminated with an excess of ribose-5-P isomerase. ASSAY B. OXIDATIONOF D-RIBITOL-5-P. The reaction mixtures contain 0.2 ml of T r i s - C 1 - E D T A buffer, 0.1 ml of D-ribitol-5-P, 0.1 ml of T P N , and 0.5 ml of ammonium acetate and enzyme in a final volume of 1 ml. The increase in absorbancy at 340 mt~ is measured after addition of the enzyme; control reaction mixtures contain all the components minus D-ribitol-5-P. Definition oJ Unit. One unit is defined as the quantity of enzyme required to catalyze the formation of 1 mieromole of product per minute. Assay B will give low values in crude extracts due to the presence of T P N H oxidase activity. 3j. Baddiley, J. G. Buchanan, B. Carss, and A. P. Mathias, J. Chem. Soc., 4583 (1956). 4The isolation of ribitol-5-P described by these authors is rather cumbersome and it can be simplified as follows: After borohydride reduction, excess borohydride is destroyed by the addition of acetaldehyde. The entire reaction mixture (starting with 700 micromoles of D-ribose-5-P) is put on a 1 × 30-cm column of Dowex 1 formate (8×) and eluted with a gradient of formic acid. The constant-volume mixing flask contains 500 ml of water, and the reservoir 3 N formic acid. The ribitol5-P is detected by phosphate analysis [P. S. Chen, Jr., T. Y. Toribara, and It. Warner, Anal. Chem. 28, 1756 (1965)] and is eluted after 450 ml of eluent has passed through the column. The ribitol-5-P is freed of formic acid by lyophilization, dissolved in water, neutralized to pH 6.0, and stored at --20 °. For identification the ribitol-5-P is hydrolyzed with E. coli alkaline phosphatase, and the ribitol is identified by paper chromatography in pyridine-ethyl acetate-H20 (2:7:1) ~ and enzymatically with ribitol dehydrogenase [J. J. Fromm, Biochim. Biophys. Acta 67, 525 (1963)1.
242
ENZYMES OF SUGAR ACTIVATION
[35]
Purification
Preparation o] Lyophilized Cells. Lactobacillus plantarum (ATCC 8014) is grown in a medium containing per liter: 4g of Difco yeast extract, 10g of Difco nutrient broth, 10g of sodium acetate, 0.2 g of MgS04-7 H20, 0.01g of NaC1, 0.01g of Fe2(S0~)3"9 H20, 0.01g of MnS04-7 H~O, and 20g of glucose. An inoculum from a stab is grown in 100 ml of this medium for 12 hours, and 6 ml of this culture is used to inoculate 3 1 of medium in a 6-1 Erlenmeyer flask; the culture is allowed to grow for 12 hours at 37 °. The cells from 18 1 of culture medium are harvested by centrifugation and washed twice with 400 ml of 0.05M Tris-C1-0.001 M EDTA, pH 8.0. The cells are finally suspended in a minimal volume of 0.05M Tris-C1-0.01 M MgC12-0.001 M EDTA, pH 8.0 and lyophilized. The lyophilized powder can be stored for at least 3 weeks at --20 ° without loss of activity. Fractionation with Protamine Sul]ate. One gram of dry cells is suspended in 8 ml of 0.05 M Tris-CI-0.01 M MgClz-0.001 M EDTA, pH 8.0, and shaken with 8 g of glass beads (0.2 mm) in a 1Nossal shaker for three 30-second periods with cooling in an ice bath between each 30-second period. To prevent foaming, a drop of capryl alcohol is added. The glass beads are allowed to settle and the supernatant fluid is centrifuged. The beads and precipitate are washed twice with 5 ml of the same buffer. The pooled supernatant fluid is fractionated with protamine sulfate. In a typical experiment as shown in the table, 18.5 ml of 2% protamine PURIFICATION OF RIBITOL-5-P DEHYDROGENASEa
Fraction Extract 1 M ammonium acetate extract of protamine precipitate 0-50% ammonium sulfate fraction pH 5.1 precipitate
Volume (ml)
Specific activity (units/rag protein × 108)
Total units
64 15
1.46 8.78
2.06 1.93
12 9
12.90 49.3
1.16 0.435
Reproduced with permission from L. Glaser, Biochim. Biophys. Acta 67, 525 (1963). sulfate is added to the enzyme and after 10 minutes the precipitate is collected by centrifugation and discarded. To the supernatant fluid an additional 16 ml of 2% protamine sulfate is added, and after 10 minutes
[36]
RIBITOL 5-PHOSPHATE DEHYDROGENASE
243
the precipitate is collected by centrifugation; the supernatant fluid is discarded. The precipitate from the second protamine addition is washed with 20 ml of 0.2M ammonium acetate (pH 7.8) and then extracted for 0.5 hours with 15 ml of 1 M ammonium acetate (pH 7.8). The insoluble residue is removed by centrifugation and discarded. To the supernatant fluid containing the enzyme is added an equal volume of saturated ammonium sulfate, and after 15 minutes the precipitate is collected by centrifugation and dissolved in 1 M ammonium acetate, pH 7.8. It is necessary to pilot the protamine step with every enzyme preparation, since the quantity of protamine which will precipitate most of the nucleic acid, but not the enzyme, varies from preparation to preparation. Acid Precipitation. The enzyme is dialyzed against 0.15M acetate buffer, pH 5.1 with stirring for 4 hours. Finally the precipitate which forms in the dialysis bag is collected by centrifugation and dissolved in 1 M ammonium acetate, pH 7.8. The enzyme can be kept frozen at all stages in the purification for at least a week without loss of activity. The summary of a typical preparation is shown in the table. The precipitation at pH 5.1 serves to remove most of the ribose-5-P isomerase, but leads to a large loss of enzyme activity. After protamine precipitation the enzyme becomes unstable except in high salt concentrations. Some batches of Lactobacillus plantarum do not yield active enzyme preparations unless the initial extraction in the Nossal shaker is also carried out in 1 M ammonium acetate pH 7.8. No further attempt has been made to purify such preparations. Properties The enzyme will use D P N H as well as T P N H as a substrate. T P N H is used in most of the assays because the apparent T P N H oxidase in crude extracts is low compared to the D P N H oxidase. The pH optimum is 7.5; the K~ for D-ribulose-5-P is 1.5 X 10-4M and for T P N H is 9 X 10~M. The enzyme will not react with glucose-6-P, fructose-6-P, ribose, ribulose, ribitol, or arabitol, and it is not inhibited by 10-" M CDP-ribitol. Several unsuccessful attempts have been made to detect this enzyme in strains of B. subtilis which contain a ribitol teichoic acid, and therefore the mode of synthesis of D-ribitol-5-P in these organisms remains to be determined.
ENZYMES OF SUGAR ACTIVATION
[36]
amplitude is a saturating function of the substrate concentration. The half-band width is the same as that for free DPNH. These data are consistent with the hypothesis that the epimerization reaction involves oxidation of the hexose residue at carbon 4 by the bound DPN to form a ketone that becomes symmetrically placed relative to the resulting D P N H so that subsequent reduction of the ketone by that D P N H can yield either a galactose or a glucose residue. The effective pH range for the enzyme is large. At pH 6.35, 7.15, 8.00, 8.50, and 9.64, the relative activities at 27 ° were 0.88, 0.90, 1.00, 1.00, and 0.79, respectively. The Km and the turnover number (k3) for UDPgalaetose under the standard assay conditions equal 1.6 X 10-4M and 5.0 X 102 sec -1, respectively. Imae, Morikawa, and Kurahashi, is using a partially purified preparation of E. coli UDP-galactose 4-epimerase, measured a K~ for UDP-glucose of 1.0 X 10-3 M at 25 ° and pH = 8.7. The values found by these workers for the equilibrium constant, the pH optimum and the K~ for UDP-galactose are in good agreement with those given above. The activation energy for catalysis of the UDPgalactose to UDP-glucose reaction in the temperature range 10-25 ° is 10.5 kcal/mole; this results in a 1.85-fold increase in rate per 10° increase in temperature. 18y. Imae, N. Morikawa, and K. Kurahashi, J. Biochem. (Tokyo) 56, 138 (1964).
[36]
Ribitol 5-Phosphate Dehydrogenase 1
By D-Ribitol 5-phosphate +
L u i s GLASER
TPN + TPNH or ~ Doribulose5-phosphate -t- or -t- H+ DPN + DPNH
Assay Method
Principle. The reaction is measured by determining the absorbanee change at 340 m~ due to the formation of TPNH in the forward direction or the disappearance of T P N H in the reverse reaction. Reagents D-Ribulose 5-P, 0.003 M, prepared by the method of I-Iorecker 2 'L. Glaser, Biochim. Biophys. Acta 67, 525 (1963). :B. L. Horecker, Vol. III [29].
[35]
RIBITOL 5-PHOSPHATE DEHYDROGENASE
241
D-Ribitel-5-P, O.02M, prepared from D-ribose-5-P by reduction with NaBH43, 4 T P N H , 0.001 M, prepared daily in 0.05 M Tris-C1, pH 8.0 T P N , 0.05 M, p H 7.0 Ammonium acetate, 1 M, adjusted to pH 7.8 by the addition of NH40H 0.05 M Tris-C1--0.001 M E D T A , p H 7.8
Procedure AssAY A. REDUCTION OF D-RIBULOSE-5-P. The reaction mixtures contain 0.2 ml of T r i s - C I - E D T A buffer, 0.1 ml of ribulose-5-P, 0.1 ml of T P N H , 0.5 ml of ammonium acetate and enzyme in a final volume of 1 ml. Control reaction mixtures contain all the components minus Dribulose-5-P. The decrease in absorbancy at 340 m~ is measured at 25 ° after addition of the enzyme. In all but the purest fractions D-ribose-5-P can replace D-ribulose-5-P since the enzyme is contaminated with an excess of ribose-5-P isomerase. ASSAY B. OXIDATIONOF D-RIBITOL-5-P. The reaction mixtures contain 0.2 ml of T r i s - C 1 - E D T A buffer, 0.1 ml of D-ribitol-5-P, 0.1 ml of T P N , and 0.5 ml of ammonium acetate and enzyme in a final volume of 1 ml. The increase in absorbancy at 340 mt~ is measured after addition of the enzyme; control reaction mixtures contain all the components minus D-ribitol-5-P. Definition oJ Unit. One unit is defined as the quantity of enzyme required to catalyze the formation of 1 mieromole of product per minute. Assay B will give low values in crude extracts due to the presence of T P N H oxidase activity. 3j. Baddiley, J. G. Buchanan, B. Carss, and A. P. Mathias, J. Chem. Soc., 4583 (1956). 4The isolation of ribitol-5-P described by these authors is rather cumbersome and it can be simplified as follows: After borohydride reduction, excess borohydride is destroyed by the addition of acetaldehyde. The entire reaction mixture (starting with 700 micromoles of D-ribose-5-P) is put on a 1 × 30-cm column of Dowex 1 formate (8×) and eluted with a gradient of formic acid. The constant-volume mixing flask contains 500 ml of water, and the reservoir 3 N formic acid. The ribitol5-P is detected by phosphate analysis [P. S. Chen, Jr., T. Y. Toribara, and It. Warner, Anal. Chem. 28, 1756 (1965)] and is eluted after 450 ml of eluent has passed through the column. The ribitol-5-P is freed of formic acid by lyophilization, dissolved in water, neutralized to pH 6.0, and stored at --20 °. For identification the ribitol-5-P is hydrolyzed with E. coli alkaline phosphatase, and the ribitol is identified by paper chromatography in pyridine-ethyl acetate-H20 (2:7:1) ~ and enzymatically with ribitol dehydrogenase [J. J. Fromm, Biochim. Biophys. Acta 67, 525 (1963)1.
242
ENZYMES OF SUGAR ACTIVATION
[35]
Purification
Preparation o] Lyophilized Cells. Lactobacillus plantarum (ATCC 8014) is grown in a medium containing per liter: 4g of Difco yeast extract, 10g of Difco nutrient broth, 10g of sodium acetate, 0.2 g of MgS04-7 H20, 0.01g of NaC1, 0.01g of Fe2(S0~)3"9 H20, 0.01g of MnS04-7 H~O, and 20g of glucose. An inoculum from a stab is grown in 100 ml of this medium for 12 hours, and 6 ml of this culture is used to inoculate 3 1 of medium in a 6-1 Erlenmeyer flask; the culture is allowed to grow for 12 hours at 37 °. The cells from 18 1 of culture medium are harvested by centrifugation and washed twice with 400 ml of 0.05M Tris-C1-0.001 M EDTA, pH 8.0. The cells are finally suspended in a minimal volume of 0.05M Tris-C1-0.01 M MgC12-0.001 M EDTA, pH 8.0 and lyophilized. The lyophilized powder can be stored for at least 3 weeks at --20 ° without loss of activity. Fractionation with Protamine Sul]ate. One gram of dry cells is suspended in 8 ml of 0.05 M Tris-CI-0.01 M MgClz-0.001 M EDTA, pH 8.0, and shaken with 8 g of glass beads (0.2 mm) in a 1Nossal shaker for three 30-second periods with cooling in an ice bath between each 30-second period. To prevent foaming, a drop of capryl alcohol is added. The glass beads are allowed to settle and the supernatant fluid is centrifuged. The beads and precipitate are washed twice with 5 ml of the same buffer. The pooled supernatant fluid is fractionated with protamine sulfate. In a typical experiment as shown in the table, 18.5 ml of 2% protamine PURIFICATION OF RIBITOL-5-P DEHYDROGENASEa
Fraction Extract 1 M ammonium acetate extract of protamine precipitate 0-50% ammonium sulfate fraction pH 5.1 precipitate
Volume (ml)
Specific activity (units/rag protein × 108)
Total units
64 15
1.46 8.78
2.06 1.93
12 9
12.90 49.3
1.16 0.435
Reproduced with permission from L. Glaser, Biochim. Biophys. Acta 67, 525 (1963). sulfate is added to the enzyme and after 10 minutes the precipitate is collected by centrifugation and discarded. To the supernatant fluid an additional 16 ml of 2% protamine sulfate is added, and after 10 minutes
[36]
RIBITOL 5-PHOSPHATE DEHYDROGENASE
243
the precipitate is collected by centrifugation; the supernatant fluid is discarded. The precipitate from the second protamine addition is washed with 20 ml of 0.2M ammonium acetate (pH 7.8) and then extracted for 0.5 hours with 15 ml of 1 M ammonium acetate (pH 7.8). The insoluble residue is removed by centrifugation and discarded. To the supernatant fluid containing the enzyme is added an equal volume of saturated ammonium sulfate, and after 15 minutes the precipitate is collected by centrifugation and dissolved in 1 M ammonium acetate, pH 7.8. It is necessary to pilot the protamine step with every enzyme preparation, since the quantity of protamine which will precipitate most of the nucleic acid, but not the enzyme, varies from preparation to preparation. Acid Precipitation. The enzyme is dialyzed against 0.15M acetate buffer, pH 5.1 with stirring for 4 hours. Finally the precipitate which forms in the dialysis bag is collected by centrifugation and dissolved in 1 M ammonium acetate, pH 7.8. The enzyme can be kept frozen at all stages in the purification for at least a week without loss of activity. The summary of a typical preparation is shown in the table. The precipitation at pH 5.1 serves to remove most of the ribose-5-P isomerase, but leads to a large loss of enzyme activity. After protamine precipitation the enzyme becomes unstable except in high salt concentrations. Some batches of Lactobacillus plantarum do not yield active enzyme preparations unless the initial extraction in the Nossal shaker is also carried out in 1 M ammonium acetate pH 7.8. No further attempt has been made to purify such preparations. Properties The enzyme will use D P N H as well as T P N H as a substrate. T P N H is used in most of the assays because the apparent T P N H oxidase in crude extracts is low compared to the D P N H oxidase. The pH optimum is 7.5; the K~ for D-ribulose-5-P is 1.5 X 10-4M and for T P N H is 9 X 10~M. The enzyme will not react with glucose-6-P, fructose-6-P, ribose, ribulose, ribitol, or arabitol, and it is not inhibited by 10-" M CDP-ribitol. Several unsuccessful attempts have been made to detect this enzyme in strains of B. subtilis which contain a ribitol teichoic acid, and therefore the mode of synthesis of D-ribitol-5-P in these organisms remains to be determined.