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[24] d -Ribulose
[24]
D-RIBULOSE
103
3-deoxyarabonate is located on a test-strip cut from the center of each chromatogram with a periodate-benzidine spray2 Areas of the chromatograms corresponding to the location of L-2-keto-3-deoxyarabonate, R / s 0.24-0.32, are cut out and eluted with water, and the eluent is concentrated under reduced pressure. L-2-Keto-3-deoxyarabonate is obtained in approximately 15% yield.
Determinations DL- and h-2-keto-3-deoxyarabonate are assayed either chemically or enzymically. The carbonyl group is assayed with 2,4-dinitrophenylhydrazine1°; the ~-keto acid function with semicarbazide11; formaldehyde TM and fl-formylpyruvate I~ are determined eolorimetrieally following periodate oxidationS4; and the lactone, which forms upon heating at 100 ° in 0.1 M HCI for 15 rain, is measured with hydroxylamine-ferric chloride. 15 The enzymic assay is based on the absorbance increase which occurs during the NADP-dependent conversion of L-2-keto-3-deoxyarabonate to a-ketoglutarate. 16 Reagents are added to a 1-ml quartz cell with a light path of 1 cm in the following order and brought to a final volume of 0.9 ml with water: 0.4 ml of K_oHPO~-KH_~PO4, pH 7.4, containing 20 m M 2-mercaptoethanol, 0.1 ml of 5 m M N A D P ÷, 0.03-0.04 unit of aldehyde dehydrogenase, and 0.01 unit of L-2-keto-3-deoxyarabonate dehydratase. After reading the initial optical density at 340 nm against a water blank, the reaction is initiated by adding 0.1 ml of a solution containing 0.01-0.2 ~mole of L-2-keto-3-deoxyarabonate, and the absorbance increase at 340 nm due to N A D P H formation is measured spectrophotometrically. The reaction is complete within 5-10 min. 9j. A. Cifonelli and F. Smith, Anal. Chem. 26, 1132 (1954). ~oH. Bohme and O. Winkler, Z. Anal. Chem. 412, 1 (1954). 11j. MacGee and M. Doudoroff, J. Biol. Chem. 210, 617 (1954). 12D. A. McFadyen, J. Biol. Chem. 158, 107 (1945). 13A. Weissbach and J. Hurwitz, d. Biol. Chem. 234, 705 (1959). 1, W. R. Frisell, L. A. Meech, and C. G. Mackenzie, J. Biol. Chem. 207, 709 (1954). ~ S. Hestrin, J. Biol. Chem. 180, 249 (1949). ~ See this series, Vol. 42 [50].
[24] D-Ribulose B y R. P. ~/IORTLOCK
D-Ribulose may be prepared by chemical or enzymic methods. Normally enzymic means of preparation yield D-ribulose possessing higher
104
PREPARATION OF SUBSTRATES
[24]
biological activity as determined by measuring the percentage of sugar utilized by a specific enzyme. Methods for the enzymic or chemical preparation of D-ribulose have been described previously. 1 The chemical method involves refluxing D-ribose with dry pyridine and then separation of the small yield of D-ribulose from excess D-ribose and contaminating sugars by means of a Dowex borate column or by preparation of the o-nitrophenyl hydrazone. The enzymic method which has been described requires the purification of ribitol dehydrogenase to catalyze the oxidation of ribitol to D-ribulose. Recently a procedure has been described utilizing whole cells of a strain of Klebsiella aerogenes to oxidize ribitol (adonitol) to D-ribulose. By this method the D-ribulose is collected in the cell-free supernatant after removal of the cells by centrifugation. The advantages of the latter method are the high yield of D-ribulose, approaching 100% of the ribitol added to the cell suspension, and the ease of recovery of the D-ribulose from the cell-free supernatant. A mutant strain of Aerobacter aerogenes which can be employed for this purpose is commercially available. 2 Principle. Klebsiella (Aerobacter) aerogenes possesses an inducible enzyme pathway for the degradation of ribitol. Ribitol dehydrogenase catalyzes the oxidation of ribitol to D-ribulose, which is then phosphorylated by D-ribulose kinase with the formation of D-ribulose 5-phosphate. Mutants constitutive for the enzymes of this pathway can be isolated by utilizing an uncommon pentitol, xylitol, as the sole carbon and energy source for growth. A further isolation of a n-ribulokinase-negative mutant results in a strain which is constitutive for ribitol dehydrogenase but is unable to grow utilizing ribitol as the carbon and energy source. Such a mutant will oxidize ribitol to D-ribulose with the accumulation of D-ribulose in the medium. Method. The mutant strain of Aerobacter aerogenes PRLR3 utilized for the production of D-ribulose is a uracil-requiring auxotroph, constitutive for ribitol dehydrogenase but negative for n-ribulose kinase activity2 The organism is grown in 200 ml of medium in a l-liter flask on a New Brunswick rotary shaker at 30 °. The salts medium consists of 1.5 g of KH2P04, 7.2 g of Na~HP04, 3.0 g of (NH4)2S04, 0.20 g of MgS04, 0.005 g of FeS04, in 1 liter of distilled water. Uracil is added to a concentration of 0.05 g per liter, and casein hydrolyzate to a concentration of 10 g per liter. Concentrated solutions of magnesium sulfate, uracil, and casein 1This series, Vol. 9 [39]. Sigma Chemical Co., St. Louis, Missouri. 3E. J. Oliver, T. M. Bisson, D. J. LeBlanc, and R. P. Mortlock, Anal. Biochem. 27, 300 (1969).
[24]
D-RIBULOSE
105
hydrolyzate are autoclaved separately and added to the basic salts medium after cooling. After inoculation of the medium, growth is at 30 ° under aerobic conditions. When growth is complete the cells are harvested by centrifugation, resuspended in distilled water to one-tenth of their original volume, collected by centrifugation a second time, and then resuspended in one-half of the original volume of sterile 5 mM phosphate buffer at pH 7.5. Ribitol is added to a concentration of 0.5%, and the cell suspension is incubated under the same aerobic conditions at 30 ° . Samples are removed at time intervals to measure D-ribulose formation by means of the cysteine-carbazole test of Dische and Borenfreund. 4 Using D-ribulose-o-nitrophenylhydrazone as a standard, ~ when the quantity of D-ribulose present is equal to the amount of ribitol initially added, or if the amount of D-ribulose present reaches a stationary value, the cells are removed by centrifugation and the cell-free supernatant is saved. If centrifugation is carried out using aseptic technique, the cell pellet may be resuspended in fresh phosphate buffer and additional ribitol added for the production of more D-ribulose. The absence of uracil during the oxidation of ribitol to D-ribulose ensures that D-ribulose kinase-posirive revertants will not be selected during this procedure. Occasionally with this strain of Aerobacter aerogenes, the percentage of ribitol converted to D-ribulose did not reach 100%, and after reaching a stationary value the amount of D-ribulose present in the medium began to decrease. It was observed that D-ribulose could be utilized by the L-fucose catabolic pathway and the enzymes of this inducible pathway were normally present at high basal levels. An additional mutation was added to the strain to make it unable to utilize L-fucose as a growth substrate. With this latter strain, yields of D-ribulose approaching 100% have been consistently obtained. In two separate experiments using 0.2% and 0.5% ribitol, respectively, over 95% of the ribitol added was converted to D-ribulose within 4 hr incubation. When the oxidation of the ribitol to D-ribulose is completed, the cells are removed by centrifugation and the cell-free supernatant concentrated to about one-fifth of its original volume by evaporation with the temperature maintained under 40 °. The solution is deionized by passage through Dowex 50 (H +) and Dowex 3 (CO~-). After further concentration by evaporation under vacuum, the preparation obtained is sufficiently pure to be utilized in most experiments requiring n-ribulose as a substrate. Traces of ribitol and D-arabinose increase with storage of *This series, Vol. 3 [12].
106
PREPARATION OF SUBSTRATES
[25]
the frozen solution. If additional purity is required, the o-nitrophenylhydrazone derivative can be prepared and crystallized as described by Cohen. 5 5S. S. Cohen, J. Biol. Chem. 201, 71 (1953).
[25] A n E n z y m i c S y n t h e s i s Y i e l d i n g C r y s t a l l i n e S o d i u m Pyruvate Labeled with Isotopic Hydrogen By H. PAUL MELOCHE CO~I C[ ~ O
H--C--H
,
H
F pyruvate /
CO~I
-]
~--O--EIH,
CO~H OH,
"C~---O
lyase
L.z\
J
.'.
Principle. Selected lyases catalyze reactions proceeding through enzyme-bound pyruvyl enolates. When such reactions are carried out in water labeled with isotopic hydrogen, an exchange reaction between the methyl protons of pyruvate and hydrogen isotope occurs. Since the three methyl hydrogens of pyruvate are symmetrical, the reaction equilibrates at the exchange of three equivalents of label into pyruvate. This article details the use of 2-keto-3-deoxy-6-P-gluconate (KDPG) aldolase 1 for such a reaction since this enzyme is fully active in a saturated pyruvate solution. 2 A mathematical treatment of initial data is presented, which allows one to predict the incubation time required to approach equilibration between the three methyl hydrogens of pyruvate and hydrogen isotope of solvent. In addition, an experiment is described showing how one converts the ratio of deuteration to tritiation into tritium and deuterium isotope effect values. General M e t h o d
Reagents Crystalline sodium pyruvate Purified pyruvate lyase (2-keto-3-deoxy-6-P-gluconate aldolase) l TOH or D20 Ethanol (95%) 1 H. P. Meloche, J. M. Ingram, and W. A. Wood, this series, Vol. 9, p. 520. 2 It. P. Meloche and Lillian Lin, Abstr. 73rd Meeting, Amer. Soc. Microbiol. 176, P 215 (1973).
D-RIBULOSE
103
3-deoxyarabonate is located on a test-strip cut from the center of each chromatogram with a periodate-benzidine spray2 Areas of the chromatograms corresponding to the location of L-2-keto-3-deoxyarabonate, R / s 0.24-0.32, are cut out and eluted with water, and the eluent is concentrated under reduced pressure. L-2-Keto-3-deoxyarabonate is obtained in approximately 15% yield.
Determinations DL- and h-2-keto-3-deoxyarabonate are assayed either chemically or enzymically. The carbonyl group is assayed with 2,4-dinitrophenylhydrazine1°; the ~-keto acid function with semicarbazide11; formaldehyde TM and fl-formylpyruvate I~ are determined eolorimetrieally following periodate oxidationS4; and the lactone, which forms upon heating at 100 ° in 0.1 M HCI for 15 rain, is measured with hydroxylamine-ferric chloride. 15 The enzymic assay is based on the absorbance increase which occurs during the NADP-dependent conversion of L-2-keto-3-deoxyarabonate to a-ketoglutarate. 16 Reagents are added to a 1-ml quartz cell with a light path of 1 cm in the following order and brought to a final volume of 0.9 ml with water: 0.4 ml of K_oHPO~-KH_~PO4, pH 7.4, containing 20 m M 2-mercaptoethanol, 0.1 ml of 5 m M N A D P ÷, 0.03-0.04 unit of aldehyde dehydrogenase, and 0.01 unit of L-2-keto-3-deoxyarabonate dehydratase. After reading the initial optical density at 340 nm against a water blank, the reaction is initiated by adding 0.1 ml of a solution containing 0.01-0.2 ~mole of L-2-keto-3-deoxyarabonate, and the absorbance increase at 340 nm due to N A D P H formation is measured spectrophotometrically. The reaction is complete within 5-10 min. 9j. A. Cifonelli and F. Smith, Anal. Chem. 26, 1132 (1954). ~oH. Bohme and O. Winkler, Z. Anal. Chem. 412, 1 (1954). 11j. MacGee and M. Doudoroff, J. Biol. Chem. 210, 617 (1954). 12D. A. McFadyen, J. Biol. Chem. 158, 107 (1945). 13A. Weissbach and J. Hurwitz, d. Biol. Chem. 234, 705 (1959). 1, W. R. Frisell, L. A. Meech, and C. G. Mackenzie, J. Biol. Chem. 207, 709 (1954). ~ S. Hestrin, J. Biol. Chem. 180, 249 (1949). ~ See this series, Vol. 42 [50].
[24] D-Ribulose B y R. P. ~/IORTLOCK
D-Ribulose may be prepared by chemical or enzymic methods. Normally enzymic means of preparation yield D-ribulose possessing higher
104
PREPARATION OF SUBSTRATES
[24]
biological activity as determined by measuring the percentage of sugar utilized by a specific enzyme. Methods for the enzymic or chemical preparation of D-ribulose have been described previously. 1 The chemical method involves refluxing D-ribose with dry pyridine and then separation of the small yield of D-ribulose from excess D-ribose and contaminating sugars by means of a Dowex borate column or by preparation of the o-nitrophenyl hydrazone. The enzymic method which has been described requires the purification of ribitol dehydrogenase to catalyze the oxidation of ribitol to D-ribulose. Recently a procedure has been described utilizing whole cells of a strain of Klebsiella aerogenes to oxidize ribitol (adonitol) to D-ribulose. By this method the D-ribulose is collected in the cell-free supernatant after removal of the cells by centrifugation. The advantages of the latter method are the high yield of D-ribulose, approaching 100% of the ribitol added to the cell suspension, and the ease of recovery of the D-ribulose from the cell-free supernatant. A mutant strain of Aerobacter aerogenes which can be employed for this purpose is commercially available. 2 Principle. Klebsiella (Aerobacter) aerogenes possesses an inducible enzyme pathway for the degradation of ribitol. Ribitol dehydrogenase catalyzes the oxidation of ribitol to D-ribulose, which is then phosphorylated by D-ribulose kinase with the formation of D-ribulose 5-phosphate. Mutants constitutive for the enzymes of this pathway can be isolated by utilizing an uncommon pentitol, xylitol, as the sole carbon and energy source for growth. A further isolation of a n-ribulokinase-negative mutant results in a strain which is constitutive for ribitol dehydrogenase but is unable to grow utilizing ribitol as the carbon and energy source. Such a mutant will oxidize ribitol to D-ribulose with the accumulation of D-ribulose in the medium. Method. The mutant strain of Aerobacter aerogenes PRLR3 utilized for the production of D-ribulose is a uracil-requiring auxotroph, constitutive for ribitol dehydrogenase but negative for n-ribulose kinase activity2 The organism is grown in 200 ml of medium in a l-liter flask on a New Brunswick rotary shaker at 30 °. The salts medium consists of 1.5 g of KH2P04, 7.2 g of Na~HP04, 3.0 g of (NH4)2S04, 0.20 g of MgS04, 0.005 g of FeS04, in 1 liter of distilled water. Uracil is added to a concentration of 0.05 g per liter, and casein hydrolyzate to a concentration of 10 g per liter. Concentrated solutions of magnesium sulfate, uracil, and casein 1This series, Vol. 9 [39]. Sigma Chemical Co., St. Louis, Missouri. 3E. J. Oliver, T. M. Bisson, D. J. LeBlanc, and R. P. Mortlock, Anal. Biochem. 27, 300 (1969).
[24]
D-RIBULOSE
105
hydrolyzate are autoclaved separately and added to the basic salts medium after cooling. After inoculation of the medium, growth is at 30 ° under aerobic conditions. When growth is complete the cells are harvested by centrifugation, resuspended in distilled water to one-tenth of their original volume, collected by centrifugation a second time, and then resuspended in one-half of the original volume of sterile 5 mM phosphate buffer at pH 7.5. Ribitol is added to a concentration of 0.5%, and the cell suspension is incubated under the same aerobic conditions at 30 ° . Samples are removed at time intervals to measure D-ribulose formation by means of the cysteine-carbazole test of Dische and Borenfreund. 4 Using D-ribulose-o-nitrophenylhydrazone as a standard, ~ when the quantity of D-ribulose present is equal to the amount of ribitol initially added, or if the amount of D-ribulose present reaches a stationary value, the cells are removed by centrifugation and the cell-free supernatant is saved. If centrifugation is carried out using aseptic technique, the cell pellet may be resuspended in fresh phosphate buffer and additional ribitol added for the production of more D-ribulose. The absence of uracil during the oxidation of ribitol to D-ribulose ensures that D-ribulose kinase-posirive revertants will not be selected during this procedure. Occasionally with this strain of Aerobacter aerogenes, the percentage of ribitol converted to D-ribulose did not reach 100%, and after reaching a stationary value the amount of D-ribulose present in the medium began to decrease. It was observed that D-ribulose could be utilized by the L-fucose catabolic pathway and the enzymes of this inducible pathway were normally present at high basal levels. An additional mutation was added to the strain to make it unable to utilize L-fucose as a growth substrate. With this latter strain, yields of D-ribulose approaching 100% have been consistently obtained. In two separate experiments using 0.2% and 0.5% ribitol, respectively, over 95% of the ribitol added was converted to D-ribulose within 4 hr incubation. When the oxidation of the ribitol to D-ribulose is completed, the cells are removed by centrifugation and the cell-free supernatant concentrated to about one-fifth of its original volume by evaporation with the temperature maintained under 40 °. The solution is deionized by passage through Dowex 50 (H +) and Dowex 3 (CO~-). After further concentration by evaporation under vacuum, the preparation obtained is sufficiently pure to be utilized in most experiments requiring n-ribulose as a substrate. Traces of ribitol and D-arabinose increase with storage of *This series, Vol. 3 [12].
106
PREPARATION OF SUBSTRATES
[25]
the frozen solution. If additional purity is required, the o-nitrophenylhydrazone derivative can be prepared and crystallized as described by Cohen. 5 5S. S. Cohen, J. Biol. Chem. 201, 71 (1953).
[25] A n E n z y m i c S y n t h e s i s Y i e l d i n g C r y s t a l l i n e S o d i u m Pyruvate Labeled with Isotopic Hydrogen By H. PAUL MELOCHE CO~I C[ ~ O
H--C--H
,
H
F pyruvate /
CO~I
-]
~--O--EIH,
CO~H OH,
"C~---O
lyase
L.z\
J
.'.
Principle. Selected lyases catalyze reactions proceeding through enzyme-bound pyruvyl enolates. When such reactions are carried out in water labeled with isotopic hydrogen, an exchange reaction between the methyl protons of pyruvate and hydrogen isotope occurs. Since the three methyl hydrogens of pyruvate are symmetrical, the reaction equilibrates at the exchange of three equivalents of label into pyruvate. This article details the use of 2-keto-3-deoxy-6-P-gluconate (KDPG) aldolase 1 for such a reaction since this enzyme is fully active in a saturated pyruvate solution. 2 A mathematical treatment of initial data is presented, which allows one to predict the incubation time required to approach equilibration between the three methyl hydrogens of pyruvate and hydrogen isotope of solvent. In addition, an experiment is described showing how one converts the ratio of deuteration to tritiation into tritium and deuterium isotope effect values. General M e t h o d
Reagents Crystalline sodium pyruvate Purified pyruvate lyase (2-keto-3-deoxy-6-P-gluconate aldolase) l TOH or D20 Ethanol (95%) 1 H. P. Meloche, J. M. Ingram, and W. A. Wood, this series, Vol. 9, p. 520. 2 It. P. Meloche and Lillian Lin, Abstr. 73rd Meeting, Amer. Soc. Microbiol. 176, P 215 (1973).