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[21b] Glucose dehydrogenases—soluble
[21b]
GLUCOSE DEHYDROGENASES--SOLUBLE
107
p-chloromercuribenzoate or iodoacetate over the pH range 5-8.5 and is very stable to photooxidation. High concentrations of ammonium ion inactivate the purified enzyme, and therefore the use of (NH4)zS04 fractionation should be restricted to treatment of crude extracts. Glucose dehydrogenase is inactivated by sulfonyl chlorides, chloro- and fluorobenzenes, or acetylating agents, but can be protected by the presence of substrate. Furthermore, neutral hydroxylamine, 0.16 M, partially reverses the inactivation due to acetic anhydride and fully reverses the inactivation produced by acetyl imidazole. The latter observation provides presumptive evidence for the presence of tyrosine at the active center of ttle enzyme. Heat Resistance. The maximal thermal stability of glucose dehydrogenase occurs at pH 6.5. The half-life of the protein at 65 ° in 0.05 imidazole buffer, pH 6.5, is 3.5 minutes2 Over the pH range 8 to 6.5, the enzyme acquires two protons from the medium and undergoes a reversible dimer-to-monomer conversion. The thermal resistance of glucose dehydrogenase at pH 6.5 increases as a second order function of the concentration of the group 1 A cations, particularly Na + and K +. The effect is reversible. The half-life of the enzyme at 85 ° in 0.5 M NaC1 is 1 minute, whereas that in 5 M NaC1 is 200 minutes. By comparison, in 0.05M imidazole buffer alone, the calculated half-life of glucose dehydrogenase is 0.04 minute.
[2 l b ]
Glucose DehydrogenasesmSoluble II. Bacterium anitratum
By JENS G. HAUGE D-Glucose--+D-gluconolactone+ 2H+ + 2eAssay Method
Principle. The reduction in optical density at 600 m/~ of 2,6-dichlorophenolindophenol (DIP) is used to measure the enzyme activity. Reagents Glucose, 0.6 M DIP solution, 1.4 micromole/ml in water Potassium phosphate, 0.1 M, pH 6.0 after 1:1 dilution Enzyme, 0.4-40 units/ml
108
DEHYDROGENASES AND OXIDASES
[21b]
Procedure. The assay mixture ingredients, 1.5 ml of buffer, 0.1 ml of the glucose solution, 0.1 ml of the D I P solution, and a volume of water equal to 1.3 ml minus the volume of enzyme to be added, are mixed in a 1-cm cuvette and equilibrated at 25 °. For routine purposes a larger portion of this assay mixture may be kept in a 25 ° waterbath ready for immediate use. A cuvette filled with water serves as the blank. The reaction is started with stirring in 1-50 t~l of enzyme solution, depending on the activity. The optical density is recorded manually at 15-second intervals. The enzyme activity may be obtained more accurately from an automatic recording of the transmission of the solution, and this also expands the range of measurable concentrations to 400 units/ml. For crude extracts, a correction should be applied for the endogenous reducing power. Definition of Unit and Specific Activity. One unit of enzyme reduces 1/~mole of D I P per minute, at an optical density of 0.6. This corresponds to a AE6oo/minute of 5.0. AE~oo/minute at this optical density is obtained either directly or alternatively from the slope of the tangent to the transmission plot in the inflection point (T, 0.375) through multiplication by the empirical factor 1.56.1 Specific activity is expressed as units per milligram of protein. The protein concentration is determined by the biuret method. 2 Purification Procedure
Medium. B. anitratum is grown with good aeration in a medium containing per liter of tap water 15 g of sodium succinate.6 tt~0, 4 g of NH4C1, 2 g of K2HP04, and 0.06 g of MgSO4.7 H~O. The pH is kept between 6.6 and 7.6, and the culture is harvested when the yield reaches 1.0-1.5 g dry weight per liter. The cell paste is frozen and stored at --20 °. Step 1. A 137-g sample of frozen cell paste is thawed and suspended in 130 ml of cold 0.1 M phosphate, pH 6. All subsequent steps are performed at 2-6 °. The suspension is passed in portions through a French pressure cell (American Instrument Company, Inc.) under 9 tons of pressure. The pressate is diluted with 45 ml of the above buffer and 200 ml of water, and whole cells and large fragments are removed by two centrifugations, 5 minutes each, at 20,000 g. The sediment is washed in 80 ml of 0.01 M phosphate, pH 6, and the second supernatant fraction is added to the first. This constitutes the crude extract. Step 2. The crude extract is centrifuged for 30 minutes at 20,000 g to remove smaller cell fragments, and then is made 1% with respect to 1j. G. Hauge, J. Biol. Chem. 239, 3630 (1964). ~A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949).
[21b]
GLUCOSE DEHYDROGEN'ASES--SOLUBLE
109
protamine sulfate. After 30 minutes of stirring, the precipitate formed is removed by centrifugation. Solid ammonium sulfate is now added in steps to create 45, 55, 58, and 70% saturation, the pH being kept at 6.1-6.3. The first two steps remove a large portion of the enzyme units in the form of particulate glucose dehydrogenase, ~,4 whereas the soluble form, to be purified here, precipitates only when the saturation exceeds 58%. The yellow precipitate formed between 58 and 70% saturation is dissolved in 0.005 M phosphate, pH 7, and dialyzed with internal stirring against the same buffer for 5 hours. The material that becomes insoluble during the dialysis is removed. The preparation is routinely frozen at this point and may be stored for shorter or longer periods with only minor losses. Step 3. The dialyzate is passed through a 2.5-g DEAE-cellulose column (0.7 meq/g) that has been equilibrated with 0.005 M phosphate, pH 7. Step 4. The DEAE-cellulose effluent is adjusted to pH 6 and passed through a 1-g CM-cellulose column (0.8 meq/g) equilibrated with 0.01 M phosphate of pH 6. The column is developed with 60 ml of a linear gradient from 0.025 M phosphate, pH 6.3 to 0.025 M phosphate, 0.1 M NaC1, pH 7.0. Before storage, the enzyme of the peak-fractions is routinely concentrated by adsorption on 30 mg CM-cellulose and elution with a small volume 0.1 M phosphate, pH 7. This purification procedure (see table) has been used a great many PURIFICATION PROCEDURE FOR Bacterium anitratum GLUCOSE DEHYDROGENASE
Fraction 1. 2. 3. 4.
Crude extract Ammonium sulfate precipitate DEAE-celluloseeffluent CM-cellulosepeak fractions
Volume (ml)
Total units
505 25 29 3.3
6200 2510 2300 820
Total Specific protein activity (mg) (units/mg) 8070 185 34 1.4
0.8 13.6 67.6 570
times by the present author and found to be dependable. The specific activity of the final product may vary somewhat, mainly reflecting the specific activity of the crude extract. The specific activity reached in the purification described above was judged to be about 90% pure. Alternatively the enzyme may be purified from the membrane frac3j. G. Hauge, Biochim. et Biophys. Acta, 45, 250 (1960). 4This volume [20a, 20b].
110
DEHYDROGENASES AND OXIDASES
[21b]
tion. An initial step with deoxycholate treatment is here included, otherwise the procedure is the same2 Properties
Stability. A variable tendency to lose some activity during frozen storage and also during the last stages of purification has been noted. This may be an expression of loss of the prosthetic group (see below). Specificity. The enzyme acts on a number of aldoses with the same maximal velocity but with different Michaelis constants. 6 The Michaelis constant for D-glucose, with the electron acceptor in excess, is 5.3 X 10-~M, that of D-xylose fiftyfold larger. L-Xylose is not attacked. Maltose, lactose, and cellobiose are attacked almost as readily as Dglucose, whereas melibiose is dehydrogenated very sluggishly, fl-DGlucose is preferred to ,a-D-glucose, but this specificity is not absolute. In addition to 2,6-dichlorophenolindophenol (K,, = 1.6 X 10-4 M) phenazine methosulfate is an efficient acceptor. Methylene blue accepts electrons at 2% of the rate of indophenol, and ferricyanide at 0.3%. Flavin or pyridine nucleotides are not measurably reduced, nor cytochrome c or triphenyltetrazolium chloride. The natural acceptor is not known. The soluble cytochrome b which accompanies the dehydrogenase during most of the purification, is however reduced by glucose via the dehydrogenase, either directly or through an intermediary carrier. Inhibitors. High concentrations of substrate and acceptor inhibit the enzyme, apparently by mutual competition. Atebrin inhibits through competition with the acceptor (K~ = 4 }(10-3M). p-Chloromercuribenzoate, arsenite, cyanide, or o-phenanthroline do not inhibit. pH Optimum. The pH optimum under standard assay conditions is 5.5. With lowered substrate and increased acceptor concentrations, the pH optimum is transposed to higher values. Absorption Spectrum. The oxidized state of the enzyme is characterized by a broad absorption band in the region of 320-390 m~, peak at 347 m~, and by a ratio, E2so:E~6o, of 1.65. On addition of glucose a sharper band appears in the near ultraviolet, with a maximum at 337 m~. At the same time the absorption below 300 mg is reduced, maximally at about 260 m~, so that the ratio, E2so:E26o, is now 1.90. The 337-mt~ band also appears with dithionite or borohydride. Through titration of the prosthetic group of the intact enzyme with glucose, the following molar extinction coefficients were found: c35,(oxidized)= 15,600; ~3a7 (reduced) =38,900; cs37(difference)=24,400; czar(difference)= 15,500. '~J. G. Hauge and P. A. Hallberg, Biochim. Biophys. Acta 81, 251 (1964). ej. G. Hauge, Biochim. Biophys. Acla 45~ 263 (1960).
[21b]
GLUCOSE DEHYDROGENASES--SOLUBLE
111
Molecular Weight and Turnover Number. Glucose titration data on an average gave an equivalent weight for the enzyme of 86,000. With a sedimentation constant S2o,w of 6.2, established in the analytical ultracentrifuge, this equivalent weight probably gives the molecular weight as well. Extrapolation of the velocities observed of substrate and acceptor concentrations which permit linear Lineweaver-Burk plots to infinite concentrations give a turnover number of 320,000 min-1. From this value and the K~ for the acceptor, the rate constant for the reaction between reduced enzyme and acceptor was calculated to be 3.3 X 107 sec-lM -1. This calculation assumes the reoxidation reaction not to be limiting at infinite acceptor concentration. Dissociation and Reactivation o] the dEnzyme. The apoenzyme has most dependably been prepared by gel filtration on Sephadex G-25 at pH 2-2.5. The apoenzyme is inactive as such and does not show the 337-m~ absorption band upon chemical reduction or addition of glucose. Apoenzyme preparations could be reactivated by addition of boiled extract or a neutralized perchloric acid extract of purified enzyme. A few microliters of apoenzyme were preincubated with a similar volume of boiled juice or perchlorate extract. Half-maximal activation was observed within 2 to 20 minutes, depending on the concentration of the reactants. With excess extract, the concentration of reactivable sites could be determined; and with excess apoenzyme, the concentration of the dissociated prosthetic group could be estimated similarly. Prosthetic Group. The prosthetic group has been purified and concentrated by chromatography on DEAE-Sephadex, and some of its spectral properties have been investigated.~ These studies indicate that it is not identical to any of the prosthetic groups or cofactors whose structure is known today. The high extinction coefficient for the bound and the free reduced group is particularly noteworthy. The cytochrome chain-bound glucose dehydrogenase of B. anitratum has been demonstrated to carry the same prosthetic group, 5 and glucose oxidizing particles from various Acetobacter and Pseudomonas species may well have it. 4 Especially interesting is the case of Rhodopseudomonas spheroides, which has been observed to require an unknown factor present in the boiled soluble fraction for the reduction of phenazine methosulfate.~ This factor was adsorbed by anion exchangers and charcoal, as is the B. anitratum factor. The Rhodopseudomonas enzyme, furthermore, could be reactivated by a boiled extract of B. anitratum. 7D. J. Niederpruem and M. Doudoroff,J. Bacteriol. 89, 697 (1965).
GLUCOSE DEHYDROGENASES--SOLUBLE
107
p-chloromercuribenzoate or iodoacetate over the pH range 5-8.5 and is very stable to photooxidation. High concentrations of ammonium ion inactivate the purified enzyme, and therefore the use of (NH4)zS04 fractionation should be restricted to treatment of crude extracts. Glucose dehydrogenase is inactivated by sulfonyl chlorides, chloro- and fluorobenzenes, or acetylating agents, but can be protected by the presence of substrate. Furthermore, neutral hydroxylamine, 0.16 M, partially reverses the inactivation due to acetic anhydride and fully reverses the inactivation produced by acetyl imidazole. The latter observation provides presumptive evidence for the presence of tyrosine at the active center of ttle enzyme. Heat Resistance. The maximal thermal stability of glucose dehydrogenase occurs at pH 6.5. The half-life of the protein at 65 ° in 0.05 imidazole buffer, pH 6.5, is 3.5 minutes2 Over the pH range 8 to 6.5, the enzyme acquires two protons from the medium and undergoes a reversible dimer-to-monomer conversion. The thermal resistance of glucose dehydrogenase at pH 6.5 increases as a second order function of the concentration of the group 1 A cations, particularly Na + and K +. The effect is reversible. The half-life of the enzyme at 85 ° in 0.5 M NaC1 is 1 minute, whereas that in 5 M NaC1 is 200 minutes. By comparison, in 0.05M imidazole buffer alone, the calculated half-life of glucose dehydrogenase is 0.04 minute.
[2 l b ]
Glucose DehydrogenasesmSoluble II. Bacterium anitratum
By JENS G. HAUGE D-Glucose--+D-gluconolactone+ 2H+ + 2eAssay Method
Principle. The reduction in optical density at 600 m/~ of 2,6-dichlorophenolindophenol (DIP) is used to measure the enzyme activity. Reagents Glucose, 0.6 M DIP solution, 1.4 micromole/ml in water Potassium phosphate, 0.1 M, pH 6.0 after 1:1 dilution Enzyme, 0.4-40 units/ml
108
DEHYDROGENASES AND OXIDASES
[21b]
Procedure. The assay mixture ingredients, 1.5 ml of buffer, 0.1 ml of the glucose solution, 0.1 ml of the D I P solution, and a volume of water equal to 1.3 ml minus the volume of enzyme to be added, are mixed in a 1-cm cuvette and equilibrated at 25 °. For routine purposes a larger portion of this assay mixture may be kept in a 25 ° waterbath ready for immediate use. A cuvette filled with water serves as the blank. The reaction is started with stirring in 1-50 t~l of enzyme solution, depending on the activity. The optical density is recorded manually at 15-second intervals. The enzyme activity may be obtained more accurately from an automatic recording of the transmission of the solution, and this also expands the range of measurable concentrations to 400 units/ml. For crude extracts, a correction should be applied for the endogenous reducing power. Definition of Unit and Specific Activity. One unit of enzyme reduces 1/~mole of D I P per minute, at an optical density of 0.6. This corresponds to a AE6oo/minute of 5.0. AE~oo/minute at this optical density is obtained either directly or alternatively from the slope of the tangent to the transmission plot in the inflection point (T, 0.375) through multiplication by the empirical factor 1.56.1 Specific activity is expressed as units per milligram of protein. The protein concentration is determined by the biuret method. 2 Purification Procedure
Medium. B. anitratum is grown with good aeration in a medium containing per liter of tap water 15 g of sodium succinate.6 tt~0, 4 g of NH4C1, 2 g of K2HP04, and 0.06 g of MgSO4.7 H~O. The pH is kept between 6.6 and 7.6, and the culture is harvested when the yield reaches 1.0-1.5 g dry weight per liter. The cell paste is frozen and stored at --20 °. Step 1. A 137-g sample of frozen cell paste is thawed and suspended in 130 ml of cold 0.1 M phosphate, pH 6. All subsequent steps are performed at 2-6 °. The suspension is passed in portions through a French pressure cell (American Instrument Company, Inc.) under 9 tons of pressure. The pressate is diluted with 45 ml of the above buffer and 200 ml of water, and whole cells and large fragments are removed by two centrifugations, 5 minutes each, at 20,000 g. The sediment is washed in 80 ml of 0.01 M phosphate, pH 6, and the second supernatant fraction is added to the first. This constitutes the crude extract. Step 2. The crude extract is centrifuged for 30 minutes at 20,000 g to remove smaller cell fragments, and then is made 1% with respect to 1j. G. Hauge, J. Biol. Chem. 239, 3630 (1964). ~A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949).
[21b]
GLUCOSE DEHYDROGEN'ASES--SOLUBLE
109
protamine sulfate. After 30 minutes of stirring, the precipitate formed is removed by centrifugation. Solid ammonium sulfate is now added in steps to create 45, 55, 58, and 70% saturation, the pH being kept at 6.1-6.3. The first two steps remove a large portion of the enzyme units in the form of particulate glucose dehydrogenase, ~,4 whereas the soluble form, to be purified here, precipitates only when the saturation exceeds 58%. The yellow precipitate formed between 58 and 70% saturation is dissolved in 0.005 M phosphate, pH 7, and dialyzed with internal stirring against the same buffer for 5 hours. The material that becomes insoluble during the dialysis is removed. The preparation is routinely frozen at this point and may be stored for shorter or longer periods with only minor losses. Step 3. The dialyzate is passed through a 2.5-g DEAE-cellulose column (0.7 meq/g) that has been equilibrated with 0.005 M phosphate, pH 7. Step 4. The DEAE-cellulose effluent is adjusted to pH 6 and passed through a 1-g CM-cellulose column (0.8 meq/g) equilibrated with 0.01 M phosphate of pH 6. The column is developed with 60 ml of a linear gradient from 0.025 M phosphate, pH 6.3 to 0.025 M phosphate, 0.1 M NaC1, pH 7.0. Before storage, the enzyme of the peak-fractions is routinely concentrated by adsorption on 30 mg CM-cellulose and elution with a small volume 0.1 M phosphate, pH 7. This purification procedure (see table) has been used a great many PURIFICATION PROCEDURE FOR Bacterium anitratum GLUCOSE DEHYDROGENASE
Fraction 1. 2. 3. 4.
Crude extract Ammonium sulfate precipitate DEAE-celluloseeffluent CM-cellulosepeak fractions
Volume (ml)
Total units
505 25 29 3.3
6200 2510 2300 820
Total Specific protein activity (mg) (units/mg) 8070 185 34 1.4
0.8 13.6 67.6 570
times by the present author and found to be dependable. The specific activity of the final product may vary somewhat, mainly reflecting the specific activity of the crude extract. The specific activity reached in the purification described above was judged to be about 90% pure. Alternatively the enzyme may be purified from the membrane frac3j. G. Hauge, Biochim. et Biophys. Acta, 45, 250 (1960). 4This volume [20a, 20b].
110
DEHYDROGENASES AND OXIDASES
[21b]
tion. An initial step with deoxycholate treatment is here included, otherwise the procedure is the same2 Properties
Stability. A variable tendency to lose some activity during frozen storage and also during the last stages of purification has been noted. This may be an expression of loss of the prosthetic group (see below). Specificity. The enzyme acts on a number of aldoses with the same maximal velocity but with different Michaelis constants. 6 The Michaelis constant for D-glucose, with the electron acceptor in excess, is 5.3 X 10-~M, that of D-xylose fiftyfold larger. L-Xylose is not attacked. Maltose, lactose, and cellobiose are attacked almost as readily as Dglucose, whereas melibiose is dehydrogenated very sluggishly, fl-DGlucose is preferred to ,a-D-glucose, but this specificity is not absolute. In addition to 2,6-dichlorophenolindophenol (K,, = 1.6 X 10-4 M) phenazine methosulfate is an efficient acceptor. Methylene blue accepts electrons at 2% of the rate of indophenol, and ferricyanide at 0.3%. Flavin or pyridine nucleotides are not measurably reduced, nor cytochrome c or triphenyltetrazolium chloride. The natural acceptor is not known. The soluble cytochrome b which accompanies the dehydrogenase during most of the purification, is however reduced by glucose via the dehydrogenase, either directly or through an intermediary carrier. Inhibitors. High concentrations of substrate and acceptor inhibit the enzyme, apparently by mutual competition. Atebrin inhibits through competition with the acceptor (K~ = 4 }(10-3M). p-Chloromercuribenzoate, arsenite, cyanide, or o-phenanthroline do not inhibit. pH Optimum. The pH optimum under standard assay conditions is 5.5. With lowered substrate and increased acceptor concentrations, the pH optimum is transposed to higher values. Absorption Spectrum. The oxidized state of the enzyme is characterized by a broad absorption band in the region of 320-390 m~, peak at 347 m~, and by a ratio, E2so:E~6o, of 1.65. On addition of glucose a sharper band appears in the near ultraviolet, with a maximum at 337 m~. At the same time the absorption below 300 mg is reduced, maximally at about 260 m~, so that the ratio, E2so:E26o, is now 1.90. The 337-mt~ band also appears with dithionite or borohydride. Through titration of the prosthetic group of the intact enzyme with glucose, the following molar extinction coefficients were found: c35,(oxidized)= 15,600; ~3a7 (reduced) =38,900; cs37(difference)=24,400; czar(difference)= 15,500. '~J. G. Hauge and P. A. Hallberg, Biochim. Biophys. Acta 81, 251 (1964). ej. G. Hauge, Biochim. Biophys. Acla 45~ 263 (1960).
[21b]
GLUCOSE DEHYDROGENASES--SOLUBLE
111
Molecular Weight and Turnover Number. Glucose titration data on an average gave an equivalent weight for the enzyme of 86,000. With a sedimentation constant S2o,w of 6.2, established in the analytical ultracentrifuge, this equivalent weight probably gives the molecular weight as well. Extrapolation of the velocities observed of substrate and acceptor concentrations which permit linear Lineweaver-Burk plots to infinite concentrations give a turnover number of 320,000 min-1. From this value and the K~ for the acceptor, the rate constant for the reaction between reduced enzyme and acceptor was calculated to be 3.3 X 107 sec-lM -1. This calculation assumes the reoxidation reaction not to be limiting at infinite acceptor concentration. Dissociation and Reactivation o] the dEnzyme. The apoenzyme has most dependably been prepared by gel filtration on Sephadex G-25 at pH 2-2.5. The apoenzyme is inactive as such and does not show the 337-m~ absorption band upon chemical reduction or addition of glucose. Apoenzyme preparations could be reactivated by addition of boiled extract or a neutralized perchloric acid extract of purified enzyme. A few microliters of apoenzyme were preincubated with a similar volume of boiled juice or perchlorate extract. Half-maximal activation was observed within 2 to 20 minutes, depending on the concentration of the reactants. With excess extract, the concentration of reactivable sites could be determined; and with excess apoenzyme, the concentration of the dissociated prosthetic group could be estimated similarly. Prosthetic Group. The prosthetic group has been purified and concentrated by chromatography on DEAE-Sephadex, and some of its spectral properties have been investigated.~ These studies indicate that it is not identical to any of the prosthetic groups or cofactors whose structure is known today. The high extinction coefficient for the bound and the free reduced group is particularly noteworthy. The cytochrome chain-bound glucose dehydrogenase of B. anitratum has been demonstrated to carry the same prosthetic group, 5 and glucose oxidizing particles from various Acetobacter and Pseudomonas species may well have it. 4 Especially interesting is the case of Rhodopseudomonas spheroides, which has been observed to require an unknown factor present in the boiled soluble fraction for the reduction of phenazine methosulfate.~ This factor was adsorbed by anion exchangers and charcoal, as is the B. anitratum factor. The Rhodopseudomonas enzyme, furthermore, could be reactivated by a boiled extract of B. anitratum. 7D. J. Niederpruem and M. Doudoroff,J. Bacteriol. 89, 697 (1965).