- Email: [email protected]
[24] d -Glucose dehydrogenase from Pseudomonas fluorescens, membrane-bound
[24]
D-GLUCOSEDEHYDROGENASEFROMPseudomonas
149
2-deoxy-D-galactose, 6; D-fucose, 2; D-glucuronic acid, 2; D-xylose, 1. L-Glucose, D-fructose, 6-D-gluconolactone, D-gluconic acid, yD-galactonolactone, dulcitol, D-arabinose, xylitol, and sucrose are not oxidized. Inhibitors. The enzyme is strongly inhibited by sodium diethyldithiocarbamate and to a lesser extent by cyanide, hydroxylamine, azide, acetate, and pyruvate. Other Properties. The enzyme has a pH optimum at 6.3 and a temperature optimum at 25°. Its Km for glucose is 4 mM and for galactose 8 mM.
[24] D - G l u c o s e D e h y d r o g e n a s e f r o m P s e u d o m o n a s fluorescens, M e m b r a n e - B o u n d By
KAZUNOBU MATSUSHITA
and MINORU A M E Y A M A
D-Glucose + acceptor---, D-glucono-3-1actone + reduced acceptor
D-Glucose dehydrogenase (EC 1.1.99.a) occurs on the outer surface of the cytoplasmic membrane of oxidative bacteria such as Pseudomonas ~ and Gluconobacter 2 species, and initiates a direct oxidation of D-glucose through an electron transport chain. The enzyme is solubilized from the membrane and further purified to a homogeneous state.
Assay Method Principle. A spectrophotometric assay at 25° measures the decrease of absorbance at 600 nm of 2,6-dichlorophenolindophenol (DCIP) mediated with phenazine methosulfate (PMS). The activity can also be measured with PMS, DCIP, Wurster's blue (WB), ferricyanide, or coenzyme Q (CoQ) as an electron acceptor. 1 Reagents D-Glucose, 1 M, in distilled water Tris-HCl, buffer, 50 mM, pH 8.75 DCIP, 6.7 mM, in distilled water PMS, 20 mM, in distilled water K. Matsushita, Y. Ohno, E. Shinagawa, O. Adachi, and M. Ameyama,Agric, Biol. Chem, 44, 1505 (1980). 2 M. A m e y a m a , E. Shinagawa, K. Matsushita, and O. Adachi, Agric. Biol. Chem. 45, 851 (1981).
METHODSIN ENZYMOLOGY,VOL.89
Copyright© 1982by AcademicPress, Inc. All rightsof reproductionin any formreserved. ISBN0-12-18198%2
150
OXIDATION-REDUCTION ENZYMES
[24]
Enzyme dissolved in 0.01 M potassium phosphate, pH 6.0, containing 5 mM MgCI2 and 1% Triton X-100 Procedure. The cuvette with 1-cm light path contains 1 ml of buffer, 0.1 ml of DCIP, 0.1 ml of PMS, 0.1 ml of glucose, enzyme solution containing less than 0.1 unit, and water in a final volume of 3 ml. A reference cuvette contains all components except D-glucose. The reaction is initiated by the addition of D-glucose. Enzyme activity is measured as the initial reduction rate of DCIP. Potassium cyanide (1 mM) or sodium azide (4 mM) must be added in the reaction mixture when enzyme activity of the membrane is measured. Definition of Unit and Specific Activity. One unit of enzyme oxidizes 1/xmol of D-glucose, or reduces 1 tzmol of DCIP, per minute at 25° under the above conditions. The extinction coefficient of DCIP at pH 8.75 is taken as 15.1 mM -1, so that 1 unit of enzyme activity correspond to a AA of 5.0 at 600 nm. Specific activity is expressed as units per milligram of protein. The protein content is determined by the modified Lowry method 3 because the sample contains Triton X-100. Source of Enzyme
Microorganisms. Many strains of Pseudomonas aeruginosa and P. fluorescens are suitable sources of D-glucose dehydrogenase. Purification of the dehydrogenase ofP. fluorescens FM-1 isolated in our laboratory is described below. Cultures. The stock culture is maintained on a potato-glycerol slant prepared as follows: Freshly sliced potato (200 g) is boiled in 1 liter of tap water and autoclaved for 10 rain at 2 kg/cm 2. The autoclaved gruel is centrifuged at 12,000 g for 20 min, and a light-yellow supernatant is obtained. To the supernatant are added dried yeast (10 g), glycerol (20 g), polypeptone (10 g), D-glucose (5 g), and agar powder (15 g) in 1 liter of tap water. The organism is grown in a synthetic medium containing 5 g of sodium o-gluconate as the sole carbon source, 1 g of ammonium sulfate, 0.75 g of K2HPO4, 0.25 g of KH~PO4, 0.3 g of MgSO4 • 7 H20, 0.03 g of FeSO4 • 7 H~O, and 0.5 g of yeast extract in 1 liter. Cultures are grown aerobically in a 50-liter jar fermentor containing 30 liters of the medium at 30° and harvested at the late exponential phase (about 7 hr). Purification Procedure a Potassium phosphate buffer (pH 6.0) containing 5 mM MgC12 is used throughout the purification process. All procedures are carried out at 0-5 ° . 3 j. R. Dulley and P. A. Grieve, Anal. Biochern. 64, 136 (1975).
[24]
D-GLUCOSEDEHYDROGENASEFROMPseudomonas
151
Step I. Preparation of Membrane Fraction. Cells are harvested by continuous-flow centrifugation at 10,000 g at <4 °, washed with distilled water, and stored at - 2 0 ° until use. About 100 g of wet cells are suspended in a sixfold volume of 0.01 M buffer, and DNase is added to 10 tzg/ml. The cell suspension is twice passed through a French press (American Instrument Co.) at 1000 kg/cm ~and centrifuged at 1800g for 10 rain to remove intact cells. The membrane fraction is collected by centrifugation at 68,000 g for 60 min and stored at - 2 0 ° until use. Step 2. Solubilization of Enzyme. The membrane fraction prepared from 100 g of wet cells is suspended in 0.01 M buffer at a final protein concentration of 10 mg/ml. Ten percent cholic acid (pH 7.4) is added to 1% concentration. After stirring for 2 hr, the suspension is centrifuged at 68,000 g for 60 rain, and the precipitate is suspended in 0.01 M buffer to the original protein concentration. Triton X-100 and KCI are added to 1% and 1 M, respectively, and the suspension is stirred overnight. The suspension is centrifuged at 68,000g for 60 min, and the supernatant containing D-glucose dehydrogenase is obtained. Step 3. Polyethylene Glycol Fractionation. Polyethylene glycol 6000 is added to the supernatant, to 18% (v/v), and the mixture is stirred for 30 min. The supernatant is obtained by centrifugation at 10,000g for 20 rain. Step 4. Ethanol Fractionation. Cold ethanol ( - 2 0 °) is added to the supernatant up to 20% (v/v), and the precipitate formed by stirring for 30 min is removed by centrifugation at 10,000 g for 20 rain. To the supernatant, ethanol is further added to 40% (v/v), and the precipitate is collected. The precipitate is suspended in 0.01 M buffer, and Triton X-100 is added to 1% in a final volume of 30 ml. The suspension is stirred overnight, then centrifuged at 10,000 g for 20 rain to obtain a clear supernatant. Step 5. Hydroxyapatite Column Chromatography. The supernatant is applied to a hydroxyapatite column (3.5 x 5 cm) preequilibrated with 0.001 M buffer containing 1% Triton X-100. The column is washed with 300 ml of the same buffer, and the enzyme is eluted with 0.05 M buffer containing 1% Triton X-100. The peak fractions are pooled as the purified enzyme, and the fractions in the tailing portion is discarded because of impurities. In case some impurities remain, especially when the membrane starting material has a specific activity of less than 2.0 units per milligram of protein, further purification can be achieved in step 6. Step 6. Phenyl-Sepharose Column Chromatography. KCI is added to 2 M in the pooled fraction, and the solution is applied to a phenyl-Sepharose CL-4B (Pharmacia Fine Chemicals) column (1 x 4 cm) washed with 0.01 M buffer containing 2 M KC1. The column is washed with 100 ml of 0.01 M buffer containing 2 M KCI. The enzyme is eluted by a linear gradient consisting of 150 ml of 0.01 M buffer-0.05% Triton X-100, and 150 ml of 0.01 M buffer-l% Triton X-100.
152
OXIDATION--REDUCTION ENZYMES
[24]
TABLE I PURIFICATION OF D-GLUCOSE DEHYDROGENASE FROM
Pseudomonas fluorescens
Fraction
Total protein (rag)
Total activity (units)
Specific activity (units/rag protein)
Yield (%)
Membrane fraction Solubilized fraction PEG a supernatant Triton supernatant Hydroxyapatite
1958 419 85 11 2.8
6814 3749 3388 2685 1080
3.5 8.9 40 244 386
100 55 50 39 16
PEG, polyethylene glycol 6000. A t y p i e a l purification is summarized in Table I. The purified D-glucose dehydrogenase is nearly homogeneous in urea-sodium dodecyl sulfate (SDS) gel electrophoresis. Properties Molecular Properties. 1,4 The e n z y m e shows a single protein band with 87,000 ± 2400 of molecular weight b y u r e a - S D S gel electrophoresis. The molecular weight o f the e n z y m e is also estimated to be 93,000 by sucrose density gradient centrifugation in the presence of Triton X-100. These findings indicate that the e n z y m e is a single polypeptide and is present as a monomer in the presence o f Triton X-100. By lowering the concentration' of Triton X-100 in the e n z y m e solution, however, the e n z y m e aggregates and the activity decreases. The lost activity returns upon addition o f detergent. This activation is also caused by adding phosphatidylglycerol, cardiolipin, or phospholipid extracted from P s e u d o m o n a s , but not phosphatidylethanolamine. Thus, D-glucose dehydrogenase may be highly hydrophobic. This fact is confirmed by amino acid analysis o f the purified enzyme, which shows a polarity o f 39.7%. Prosthetic Group. 5 The absorption spectrum o f the purified enzyme, with the Triton X-100 replaced by Brij 58, shows a peak at 285 nm and a shoulder at 275 nm. By the addition o f D-glucose or sodium borohydride, an absorption peak at 340 nm appears owing to reduction o f the prosthetic
* K. Matsushita, Y. Ohno, E. Shinagawa, O. Adachi, and M. Ameyama, Agric. Biol. Chem. 416, 1007 (1982). 5 M. Ameyama, K. Matsushita, Y. Ohno, E. Shinagawa, and O. Adachi, FEBS Lett. 130, 179 (1981).
[24]
D-GLUCOSE DEHYDROGENASE FROM P s e u d o m o n a s
153
T A B L E II KINETIC PARAMETERS OF D-GLuCOSE DEHYDROGENASE FOR VARIOUS ELECTRON ACCEPTORS
Km Electron acceptors ~
(raM)
Ymaxb
DCIP Ferricyanide WB PMS CoQ1 CoQ2 d CoQ4 a CoQnd
1.60 0.69 0.56 0,13 0.060 0.061 0.031 0.0097
1220 57 290 258 24 28 5.7 2.0
Optimum p H (6.0) c 4.5 8.75 8.75 6.5 ----
a DCIP, dichlorophenolindophenol; W B , Wurster's blue; PMS, phenazine methosulfate; CoQ, coenzyme Q. b Micromoles of D-glucose oxidized per minute per milligram of enzyme protein. c It is impossible to assay below pH 5.5. The assay was performed at pH 6.0. d The reaction mixture contains 5% ethanol.
group. The prosthetic group extracted from the purified enzyme shows an absorption spectrum having a peak at 246 nm and a shoulder at 326 nm. The peak at 246 nm is decreased and that at 326 nm is increased by the addition of sodium borohydride. The prosthetic group has a fluorescence maximum at 480 nm with excitation at 370 nm and excitation maxima at 370 nm, 330 nm, and 260 nm with emission at 480 nm. These characteristics indicate that the prosthetic group of the enzyme is pyrroloquinoline quinone, which is isolated from cells of methylotrophic bacteria 6 and has been shown to be a prosthetic group of both methanol dehydrogenase of tlyphomicrobium X and the D-glucose dehydrogenase of Acinetobacter calcoaceticus. 7 The same prosthetic group is found also in D-glucose dehydrogenase o f Gluconobacter, 2 alcohol dehydrogenase of acetic acid bacteria (see this volume [76]), and aldehyde dehydrogenase of acetic acid bacteria (see this volume [82]). Kinetic Properties. 1.4 r~-Glucose dehydrogenase has a dual pH optimum depending on the electron acceptor used. The enzyme has an acidic optimum pH with DCIP or ferricyanide, and the activity with PMS or WB shows a maximum at pH 8.75. The Michaelis constant for D-glucose is S. A. Salisbury, H. S. Forrest, W. B. T. Crure, and O, K e n n a r d , Nature (London) 280, 843 (1979). 7 j. A. Duine, J. Frank, Jr., and J. K. Van Zeeland, FEBS Lett. 108, 443 (1979).
154
OXIDATION--REDUCTION ENZYMES
[25]
also different depending on pH: 0.47 mM at pH 6.0 with DCIP and 6.3 mM at pH 8.75 with PMS-DCIP. Kinetic parameters of the purified enzyme for various electron acceptors are shown in Table II. CoQ has the highest affinity for the enzyme. CoQ and its long-chain homologs are directly reduced by the enzyme. Pseudomonas species have CoQ9 as a natural ubiquinone, and at a slower but significant rate the enzyme is able to reduce CoQ9 solubilized with 5% ethanol or phospholipid vesicles. The enzyme activity is completely inhibited with p-benzoquinone (1.7 mM) or EDTA (3.3 mM). Substrate Specificity and Reaction Product. 1 D-Glucose dehydrogenase possesses a fairly broad substrate specificity as follows: p-glucose, 100%; o-xylose, 13%; o-mannose, 8.6%; L-rhamnose, 7.5%; o-galactose, 6.5%; maltose, 3.2%; and L-arabinose, 2.8%. The enzyme does not oxidize p-fructose, a-sorbose, D-arabinose, a-methylglucoside, o-ribose, sucrose, D-gluconate, and D-galactonate. A reaction product of the enzyme is D-glucono-8-1actone. Immunological Properties. Antibody elicited with the purified p-glucose dehydrogenase shows a single or diffused precipitin line against the purified enzyme in a Ouchterlony double-diffusion analysis. Immunoprecipitates between the antibody and the purified enzyme or membrane-solubilized supernatant show only one polypeptide band in addition to the T-globulin subunits in urea-SDS gel electrophoresis. The antibody inhibits the activity of the purified enzyme, but not that of the membrane.
[25] D - F r u c t o s e D e h y d r o g e n a s e f r o m Gluconobacter industrius, M e m b r a n e - B o u n d
By MINORU AMEYAMA and OSAO ADAC~I D-Fructose + acceptor~ 5-keto-D-fructose + reduced acceptor D-Fructose dehydrogenase (EC 1.1.99.11) occurs on the outer surface of the cytoplasmic membrane of Gluconobacter species. The enzyme is solubilized from the membrane and further purified to a homogeneous state.
Assay Method Principle. The reaction rate can be estimated (a) by spectrophotometry in the presence of 2,6-dichlorophenolindophenol and phena-
METHODS IN ENZYMOLOGY, VOL. 89
Copyright © 1982by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181989-2
D-GLUCOSEDEHYDROGENASEFROMPseudomonas
149
2-deoxy-D-galactose, 6; D-fucose, 2; D-glucuronic acid, 2; D-xylose, 1. L-Glucose, D-fructose, 6-D-gluconolactone, D-gluconic acid, yD-galactonolactone, dulcitol, D-arabinose, xylitol, and sucrose are not oxidized. Inhibitors. The enzyme is strongly inhibited by sodium diethyldithiocarbamate and to a lesser extent by cyanide, hydroxylamine, azide, acetate, and pyruvate. Other Properties. The enzyme has a pH optimum at 6.3 and a temperature optimum at 25°. Its Km for glucose is 4 mM and for galactose 8 mM.
[24] D - G l u c o s e D e h y d r o g e n a s e f r o m P s e u d o m o n a s fluorescens, M e m b r a n e - B o u n d By
KAZUNOBU MATSUSHITA
and MINORU A M E Y A M A
D-Glucose + acceptor---, D-glucono-3-1actone + reduced acceptor
D-Glucose dehydrogenase (EC 1.1.99.a) occurs on the outer surface of the cytoplasmic membrane of oxidative bacteria such as Pseudomonas ~ and Gluconobacter 2 species, and initiates a direct oxidation of D-glucose through an electron transport chain. The enzyme is solubilized from the membrane and further purified to a homogeneous state.
Assay Method Principle. A spectrophotometric assay at 25° measures the decrease of absorbance at 600 nm of 2,6-dichlorophenolindophenol (DCIP) mediated with phenazine methosulfate (PMS). The activity can also be measured with PMS, DCIP, Wurster's blue (WB), ferricyanide, or coenzyme Q (CoQ) as an electron acceptor. 1 Reagents D-Glucose, 1 M, in distilled water Tris-HCl, buffer, 50 mM, pH 8.75 DCIP, 6.7 mM, in distilled water PMS, 20 mM, in distilled water K. Matsushita, Y. Ohno, E. Shinagawa, O. Adachi, and M. Ameyama,Agric, Biol. Chem, 44, 1505 (1980). 2 M. A m e y a m a , E. Shinagawa, K. Matsushita, and O. Adachi, Agric. Biol. Chem. 45, 851 (1981).
METHODSIN ENZYMOLOGY,VOL.89
Copyright© 1982by AcademicPress, Inc. All rightsof reproductionin any formreserved. ISBN0-12-18198%2
150
OXIDATION-REDUCTION ENZYMES
[24]
Enzyme dissolved in 0.01 M potassium phosphate, pH 6.0, containing 5 mM MgCI2 and 1% Triton X-100 Procedure. The cuvette with 1-cm light path contains 1 ml of buffer, 0.1 ml of DCIP, 0.1 ml of PMS, 0.1 ml of glucose, enzyme solution containing less than 0.1 unit, and water in a final volume of 3 ml. A reference cuvette contains all components except D-glucose. The reaction is initiated by the addition of D-glucose. Enzyme activity is measured as the initial reduction rate of DCIP. Potassium cyanide (1 mM) or sodium azide (4 mM) must be added in the reaction mixture when enzyme activity of the membrane is measured. Definition of Unit and Specific Activity. One unit of enzyme oxidizes 1/xmol of D-glucose, or reduces 1 tzmol of DCIP, per minute at 25° under the above conditions. The extinction coefficient of DCIP at pH 8.75 is taken as 15.1 mM -1, so that 1 unit of enzyme activity correspond to a AA of 5.0 at 600 nm. Specific activity is expressed as units per milligram of protein. The protein content is determined by the modified Lowry method 3 because the sample contains Triton X-100. Source of Enzyme
Microorganisms. Many strains of Pseudomonas aeruginosa and P. fluorescens are suitable sources of D-glucose dehydrogenase. Purification of the dehydrogenase ofP. fluorescens FM-1 isolated in our laboratory is described below. Cultures. The stock culture is maintained on a potato-glycerol slant prepared as follows: Freshly sliced potato (200 g) is boiled in 1 liter of tap water and autoclaved for 10 rain at 2 kg/cm 2. The autoclaved gruel is centrifuged at 12,000 g for 20 min, and a light-yellow supernatant is obtained. To the supernatant are added dried yeast (10 g), glycerol (20 g), polypeptone (10 g), D-glucose (5 g), and agar powder (15 g) in 1 liter of tap water. The organism is grown in a synthetic medium containing 5 g of sodium o-gluconate as the sole carbon source, 1 g of ammonium sulfate, 0.75 g of K2HPO4, 0.25 g of KH~PO4, 0.3 g of MgSO4 • 7 H20, 0.03 g of FeSO4 • 7 H~O, and 0.5 g of yeast extract in 1 liter. Cultures are grown aerobically in a 50-liter jar fermentor containing 30 liters of the medium at 30° and harvested at the late exponential phase (about 7 hr). Purification Procedure a Potassium phosphate buffer (pH 6.0) containing 5 mM MgC12 is used throughout the purification process. All procedures are carried out at 0-5 ° . 3 j. R. Dulley and P. A. Grieve, Anal. Biochern. 64, 136 (1975).
[24]
D-GLUCOSEDEHYDROGENASEFROMPseudomonas
151
Step I. Preparation of Membrane Fraction. Cells are harvested by continuous-flow centrifugation at 10,000 g at <4 °, washed with distilled water, and stored at - 2 0 ° until use. About 100 g of wet cells are suspended in a sixfold volume of 0.01 M buffer, and DNase is added to 10 tzg/ml. The cell suspension is twice passed through a French press (American Instrument Co.) at 1000 kg/cm ~and centrifuged at 1800g for 10 rain to remove intact cells. The membrane fraction is collected by centrifugation at 68,000 g for 60 min and stored at - 2 0 ° until use. Step 2. Solubilization of Enzyme. The membrane fraction prepared from 100 g of wet cells is suspended in 0.01 M buffer at a final protein concentration of 10 mg/ml. Ten percent cholic acid (pH 7.4) is added to 1% concentration. After stirring for 2 hr, the suspension is centrifuged at 68,000 g for 60 rain, and the precipitate is suspended in 0.01 M buffer to the original protein concentration. Triton X-100 and KCI are added to 1% and 1 M, respectively, and the suspension is stirred overnight. The suspension is centrifuged at 68,000g for 60 min, and the supernatant containing D-glucose dehydrogenase is obtained. Step 3. Polyethylene Glycol Fractionation. Polyethylene glycol 6000 is added to the supernatant, to 18% (v/v), and the mixture is stirred for 30 min. The supernatant is obtained by centrifugation at 10,000g for 20 rain. Step 4. Ethanol Fractionation. Cold ethanol ( - 2 0 °) is added to the supernatant up to 20% (v/v), and the precipitate formed by stirring for 30 min is removed by centrifugation at 10,000 g for 20 rain. To the supernatant, ethanol is further added to 40% (v/v), and the precipitate is collected. The precipitate is suspended in 0.01 M buffer, and Triton X-100 is added to 1% in a final volume of 30 ml. The suspension is stirred overnight, then centrifuged at 10,000 g for 20 rain to obtain a clear supernatant. Step 5. Hydroxyapatite Column Chromatography. The supernatant is applied to a hydroxyapatite column (3.5 x 5 cm) preequilibrated with 0.001 M buffer containing 1% Triton X-100. The column is washed with 300 ml of the same buffer, and the enzyme is eluted with 0.05 M buffer containing 1% Triton X-100. The peak fractions are pooled as the purified enzyme, and the fractions in the tailing portion is discarded because of impurities. In case some impurities remain, especially when the membrane starting material has a specific activity of less than 2.0 units per milligram of protein, further purification can be achieved in step 6. Step 6. Phenyl-Sepharose Column Chromatography. KCI is added to 2 M in the pooled fraction, and the solution is applied to a phenyl-Sepharose CL-4B (Pharmacia Fine Chemicals) column (1 x 4 cm) washed with 0.01 M buffer containing 2 M KC1. The column is washed with 100 ml of 0.01 M buffer containing 2 M KCI. The enzyme is eluted by a linear gradient consisting of 150 ml of 0.01 M buffer-0.05% Triton X-100, and 150 ml of 0.01 M buffer-l% Triton X-100.
152
OXIDATION--REDUCTION ENZYMES
[24]
TABLE I PURIFICATION OF D-GLUCOSE DEHYDROGENASE FROM
Pseudomonas fluorescens
Fraction
Total protein (rag)
Total activity (units)
Specific activity (units/rag protein)
Yield (%)
Membrane fraction Solubilized fraction PEG a supernatant Triton supernatant Hydroxyapatite
1958 419 85 11 2.8
6814 3749 3388 2685 1080
3.5 8.9 40 244 386
100 55 50 39 16
PEG, polyethylene glycol 6000. A t y p i e a l purification is summarized in Table I. The purified D-glucose dehydrogenase is nearly homogeneous in urea-sodium dodecyl sulfate (SDS) gel electrophoresis. Properties Molecular Properties. 1,4 The e n z y m e shows a single protein band with 87,000 ± 2400 of molecular weight b y u r e a - S D S gel electrophoresis. The molecular weight o f the e n z y m e is also estimated to be 93,000 by sucrose density gradient centrifugation in the presence of Triton X-100. These findings indicate that the e n z y m e is a single polypeptide and is present as a monomer in the presence o f Triton X-100. By lowering the concentration' of Triton X-100 in the e n z y m e solution, however, the e n z y m e aggregates and the activity decreases. The lost activity returns upon addition o f detergent. This activation is also caused by adding phosphatidylglycerol, cardiolipin, or phospholipid extracted from P s e u d o m o n a s , but not phosphatidylethanolamine. Thus, D-glucose dehydrogenase may be highly hydrophobic. This fact is confirmed by amino acid analysis o f the purified enzyme, which shows a polarity o f 39.7%. Prosthetic Group. 5 The absorption spectrum o f the purified enzyme, with the Triton X-100 replaced by Brij 58, shows a peak at 285 nm and a shoulder at 275 nm. By the addition o f D-glucose or sodium borohydride, an absorption peak at 340 nm appears owing to reduction o f the prosthetic
* K. Matsushita, Y. Ohno, E. Shinagawa, O. Adachi, and M. Ameyama, Agric. Biol. Chem. 416, 1007 (1982). 5 M. Ameyama, K. Matsushita, Y. Ohno, E. Shinagawa, and O. Adachi, FEBS Lett. 130, 179 (1981).
[24]
D-GLUCOSE DEHYDROGENASE FROM P s e u d o m o n a s
153
T A B L E II KINETIC PARAMETERS OF D-GLuCOSE DEHYDROGENASE FOR VARIOUS ELECTRON ACCEPTORS
Km Electron acceptors ~
(raM)
Ymaxb
DCIP Ferricyanide WB PMS CoQ1 CoQ2 d CoQ4 a CoQnd
1.60 0.69 0.56 0,13 0.060 0.061 0.031 0.0097
1220 57 290 258 24 28 5.7 2.0
Optimum p H (6.0) c 4.5 8.75 8.75 6.5 ----
a DCIP, dichlorophenolindophenol; W B , Wurster's blue; PMS, phenazine methosulfate; CoQ, coenzyme Q. b Micromoles of D-glucose oxidized per minute per milligram of enzyme protein. c It is impossible to assay below pH 5.5. The assay was performed at pH 6.0. d The reaction mixture contains 5% ethanol.
group. The prosthetic group extracted from the purified enzyme shows an absorption spectrum having a peak at 246 nm and a shoulder at 326 nm. The peak at 246 nm is decreased and that at 326 nm is increased by the addition of sodium borohydride. The prosthetic group has a fluorescence maximum at 480 nm with excitation at 370 nm and excitation maxima at 370 nm, 330 nm, and 260 nm with emission at 480 nm. These characteristics indicate that the prosthetic group of the enzyme is pyrroloquinoline quinone, which is isolated from cells of methylotrophic bacteria 6 and has been shown to be a prosthetic group of both methanol dehydrogenase of tlyphomicrobium X and the D-glucose dehydrogenase of Acinetobacter calcoaceticus. 7 The same prosthetic group is found also in D-glucose dehydrogenase o f Gluconobacter, 2 alcohol dehydrogenase of acetic acid bacteria (see this volume [76]), and aldehyde dehydrogenase of acetic acid bacteria (see this volume [82]). Kinetic Properties. 1.4 r~-Glucose dehydrogenase has a dual pH optimum depending on the electron acceptor used. The enzyme has an acidic optimum pH with DCIP or ferricyanide, and the activity with PMS or WB shows a maximum at pH 8.75. The Michaelis constant for D-glucose is S. A. Salisbury, H. S. Forrest, W. B. T. Crure, and O, K e n n a r d , Nature (London) 280, 843 (1979). 7 j. A. Duine, J. Frank, Jr., and J. K. Van Zeeland, FEBS Lett. 108, 443 (1979).
154
OXIDATION--REDUCTION ENZYMES
[25]
also different depending on pH: 0.47 mM at pH 6.0 with DCIP and 6.3 mM at pH 8.75 with PMS-DCIP. Kinetic parameters of the purified enzyme for various electron acceptors are shown in Table II. CoQ has the highest affinity for the enzyme. CoQ and its long-chain homologs are directly reduced by the enzyme. Pseudomonas species have CoQ9 as a natural ubiquinone, and at a slower but significant rate the enzyme is able to reduce CoQ9 solubilized with 5% ethanol or phospholipid vesicles. The enzyme activity is completely inhibited with p-benzoquinone (1.7 mM) or EDTA (3.3 mM). Substrate Specificity and Reaction Product. 1 D-Glucose dehydrogenase possesses a fairly broad substrate specificity as follows: p-glucose, 100%; o-xylose, 13%; o-mannose, 8.6%; L-rhamnose, 7.5%; o-galactose, 6.5%; maltose, 3.2%; and L-arabinose, 2.8%. The enzyme does not oxidize p-fructose, a-sorbose, D-arabinose, a-methylglucoside, o-ribose, sucrose, D-gluconate, and D-galactonate. A reaction product of the enzyme is D-glucono-8-1actone. Immunological Properties. Antibody elicited with the purified p-glucose dehydrogenase shows a single or diffused precipitin line against the purified enzyme in a Ouchterlony double-diffusion analysis. Immunoprecipitates between the antibody and the purified enzyme or membrane-solubilized supernatant show only one polypeptide band in addition to the T-globulin subunits in urea-SDS gel electrophoresis. The antibody inhibits the activity of the purified enzyme, but not that of the membrane.
[25] D - F r u c t o s e D e h y d r o g e n a s e f r o m Gluconobacter industrius, M e m b r a n e - B o u n d
By MINORU AMEYAMA and OSAO ADAC~I D-Fructose + acceptor~ 5-keto-D-fructose + reduced acceptor D-Fructose dehydrogenase (EC 1.1.99.11) occurs on the outer surface of the cytoplasmic membrane of Gluconobacter species. The enzyme is solubilized from the membrane and further purified to a homogeneous state.
Assay Method Principle. The reaction rate can be estimated (a) by spectrophotometry in the presence of 2,6-dichlorophenolindophenol and phena-
METHODS IN ENZYMOLOGY, VOL. 89
Copyright © 1982by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181989-2