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[36] Hexopyranoside: Cytochrome c oxidoreductase from Agrobacterium
[36]
A 3-KETO SUGAR-FORMING ENZYME
153
Properties'
Substrate Specificity. Of 55 compounds tested as possible substrates, the only six that are oxidized by this enzyme are those that possess the L-arabino configuration and their deoxy derivatives. These are L-arabinose, D-galactose, 6-deoxy-D-galactose (n-fucose), 2-deoxy-D-galactose, 3,6-dideoxy-D-galactose (abequose), and 6-iodo-6-deoxy-D-galactose. Compounds that do not serve as substrates at 33 mM concentrations are: D-glucose, L-glucose, D-mannose, L-mannose, D-altrose, D-allose, L-galacrose, L-fucose, L-rhamnose, 2-deoxy-D-glucose, 6-deoxy-D-glucose, 6-deoxy-D-allose, D-glucuronic acid, D-galacturonic acid, D-glucose 6-phosphate, D-galactose 6-phosphate, D-glucosamine, N-acetyl-D-glucosamine, methyl ~-D-glucoside, 2-acetamido-D-allose, 2-acetamido-n-altrose, D-fructose, L-fructose, L-sorbose, D-fructose 6-phosphate, D-xylose, L-xylose, D-lyxose, D-ribose, 2-deoxy-D-ribose, D-arabinose, D-xylulose, Da-glyceraldehyde, D-mannitol, D-glucitol, myo-inositol, L-arabitol, D-arabitol, xylitol, ribitoi, maltose, cellobiose, sucrose, lactose, melezitose, turanose, melibiose, trehalose, and raffinose. Nucleotide Specificity. Both NAD and NADP serve as coenzymes. Km values at pH 8.1 are 15 ~M for NAD + and 68 ~M for NADP +. E:qect of pH and Substrate Concentration on Reaction Velocity. At high substrate concentrations the reaction velocity is maximal at about pH 9.4 and about 40% maximal at pH 8.1. However, the affinity of the enzyme for its substrate is greater at the lower pH. The respective apparent Km values for D-fucose, D-galactose, and L-arabinose are 0.50, 0.17, and 0.14 mM at pH 8.1, and 6.6, 2.0, and 0.52 mM at pH 9.4. Stability. Sephadex G-200 fractions (in l0 mM sodium phosphate buffer, pH 7.0) are stable to freezing for at least 2 months.
[36] H e x o p y r a n o s i d e : C y t o c h r o m e c O x i d o r e d u c t a s e from Agrobacterium 1 By J. VAN BEEUMEN and J. DE LEY Itexopyranoside -~ acceptor --+ 3-keto hexopyranoside -{- reduced acceptor Assay Method
Principle. Enzyme activity can be followed either manometrically as oxygen consumption, using 5-methyl phenazinium methylsulfate (PMS) 1j. Van Beeumen and J. De Ley, Eur. J. Biochem. 6, 311"(1968).
154
OXID&TION--REDUCTION ENZYMES
[36]
as intermediate electron carrier, or spectrophotometrically, as decrease in optical density at 600 nm using 2,6-dichlorophenolindophenol (DIP) as terminal acceptor. The former assay is laborious but offers the possibility to analyze the reaction products. A brief description is given in another article. 2 The latter assay is quicker and is described below.
Reagents D I P , 0.885 m M in 0.4 M phosphate buffer, pH 6.0 Lactose, 0.1 M Water, distilled Enzyme, 0.002-0.05 unit/ml
Procedure. The reaction can be followed in any spectrophotometer at 600 nm. A cuvette of 1 cm light path receives 2.58 ml of D I P solution, 0.05-0.1 ml of enzyme, and water to a final volume of 2.7 ml. The amount of enzyme used depends on the possibilities of chart speed and scale expansion of the recorder. The reaction mixture is placed in the thermostated cuvette holder at 30 ° and, after 2 min of thermal equilibration, the endogenous reaction is recorded for 1 min. Enzyme activity is measured as the initial reduction rate of D I P after mixing in quickly 0.3 ml of lactose solution, kept at 30 °. When the endogenous activity is too high, a blank is included with 0.3 ml of water instead of the substrate. Since D I P is known to be a very good acceptor with many other enzymes, one should identify the 3-keto sugar (synonym: 3-ulose) formed, as described below, whenever the presence of a 3-ulose-forming enzyme is suspected in organisms other than Agrobacterium. Definition o] Unit. One unit of enzyme is the amount that oxidizes 1 ~mole of lactose per minute. Our sample of D I P having an extinction coefficient Cme = 11.85 cm -1 mmole -1 at pH 6.0, this amount of enzyme gives a decrease of 4 optical density units under the conditions of the assay. Specific activity is expressed as number of units per milligram of protein. Source o] Enzyme. The enzyme is inducible and is known to occur only in most strains of Agrobacterium tumefaciens and A. radiobacter. The enzyme was completely purified from cells of A. tume]aciens ATCC 143.1 Less pure preparations have been reported from A. tumefacien~ strain B6, 3 BNV6, a and IAM 1525. 4 The latter strain is derived from 2j. Van Beeumen and J. De Ley, this volume [3]. E. E. Grebner, E. Kovach, and D. S. Feingold, this series, Vol. 9 [17]. 4K. Hayano and S. Fukui, J. Biol. Chem. 242, 3665 (1967).
[36]
A 3-KETO SUGAR-FORMING ENZYME
155
strain ATCC 4452. The enzyme has also been called D-aldohexopyranoside dehydrogenase3,5 or D-glucoside 3-dehydrogenase2 Purification Procedure
Culture Conditions. We purified the enzyme starting from a 150-1iter culture, but the amounts can be scaled down if required. Agrobacterium tume]aciens ATCC 143 is grown for 40 hr at 30 ° in a culture containing 1% yeast extract, 0.1% KH2PO4, 0.2% (NH4)2S04, 0.025% MgSO~'7H20, and 1% lactose, final pH 6.8. Lactose is autoclaved separately in concentrated solution. Growth is started with a 5% inoculum. Good aeration (8 liters of air per hour liter of medium) and vigorous stirring are necessary. Approximately 2 kg of cells are harvested by centrifugation at the end of the logarithmic growth phase. They are washed three times with 10 mM phosphate buffer, pH 7.0. The following steps should be carried out at a temperature not exceeding 5 °. Step 1. Preparation of Particle-Free Extract. Portions of 35 g of washed cells in 70 ml of 10 mM phosphate buffer, pH 7.0, are disrupted in a Raytheon 10 kc sonic oscillator after replacement of the air in the cup by hydrogen. Unbroken cells and debris, removed by centrifugation, are resuspended, sonicated, and centrifuged again. The particles in the pooled supernatants are spun down for 1 hr at 100,000 g for 4 hr at 40,000 g. Step 2. Removal o] Nucleic Acids. To the particle-free supernatant, 0.05 volume of a 1 M MnCl., solution is added under stirring. After 30 rain the precipitate is discarded by centrifugation at 10,000 g. The supernatant is dialyzed against a 1000-fold excess of 10 mM phosphate buffer, pH 6.9. A heavy precipitate formed during dialysis is removed by centrifugation. Step 3. Ammonium Sul]ate Fractionation. To each liter of supernarant, 288 g of solid ammonium sulfate is added, and the precipitate formed is removed by centrifugation. The supernatant is then brought to 60% saturation by the addition of 61 g of solid ammonium sulfate to each liter. The precipitate is dissolved in 100 ml of 10 mM phosphate buffer, pH 6.9, and the residual salt is removed by three dialysis experiments against 5 liters of the same buffer. Step 4. DEAE-Cellulose Chromatography. The preceding preparation is chromatographed on a 4.1 X 68 cm column with 100 g of DEAE-cellulose (0.69 meq/g) equilibrated with 10 mM phosphate buffer, pH 6.9. E. E. Grebner and D. S. Feingold,Biochera. Biophys. Res. Commun. 19, 37 (1965). 6M J. Bernaerts and J. De Ley, J. Gen. Microbiol. 22, 137 (1960).
156
OXIDATION--REDUCTION ENZYMES
[36]
The ion exchange is pretreated in batch experiments with 0.2 N NaOH, washed with distilled water, charged with 0.1 M phosphate buffer, pH 6.9, until the effluent has the same pH, and equilibrated with the same buffer, diluted 10-fold. The enzyme is eluted under 1 meter of hydrostatic pressure in the 0.23-0.28 M KC1 fraction of a 3.4-liter linear gradient of 0 to 0.4 M KC1 in 10 mM phosphate buffer, pH 6.9. The enzyme-containing fractions are reduced in volume by precipitation with (NH4)2S04 and dialyzed as described at the end of step 3. Step 5. Column Electrophoresis on Agarose. The data for the electrophoretic purification step in the table were obtained after two subsequent zone electrophoretic runs on a large LKB 5801 Porath column.1 The following experiment on a smaller LKB 3340 column (3 }( 41 cm) gives the same purification factor and a slightly better yield (85%). The column is filled with 0.18% agarose in 33 mM phosphate buffer, pH 7.27. About 30 mg of protein in 3.5 ml is applied. Electrophoresis is continued for 38 hr at 250 V and 37-38 mA. Electrode buffer is renewed after 18 hr. At the end of the run, the gel column is lifted by hydrostatic pressure (15 cm) and collected in 2-ml fractions. Enzyme activity is measured in the presence of agarose. The agarose is centrifuged at 10,000 g for 20 min, rcsuspended in buffer and centrifuged again. The enzyme is prepared for step 6 by (NH~)~S04 precipitation and dialysis against 30 mM phosphate buffer, pH 7.17. Step 6. Gel Filtration. About 40 mg of protein in 4 ml of buffer are loaded on top of a 2.5 X 71 cm column of Sephadex G-75. Eluted at 25 ml/hour, the pure enzyme is collected as the first of two well separated protein peaks. The product is pooled, concentrated with (NH4)~S04, dialyzed against 50 mM phosphate buffer, pH 6.9, and stored under nitrogen at --18 ° when not in use. The purification procedure is summarized in the table.
Properties Stability. The enzyme becomes increasingly labile upon purification, perhaps owing to gradual loss of FAD. The activity half-life of pure enzyme is about a week when kept in the deep freezer. The enzyme is stable for 2 weeks under nitrogen at --15% The best pH range is 6.5-7.4. 20 % Physical and Chemical Properties. The sedimentation coefficient s0.75 of the oxidoreductase is 5.1 S. Its molecular weight is 85,000 __ 7700, as determined with Archibald's method, and assuming a partial specific volume ~ of 0.725. The molecular weight from gel filtration on Sephadex G-200 is about 15% smaller, probably because of a slight retention of the enzyme on the substratelike dextran matrix of the gel. FAD is the
[36]
157
A 3 - K E T O S U G A R - F O R M I N G ENZYME PURIFICATION OF HEXOPYRANOSIDE:CYTOCHROME C OXIDOREDUCTASE a
Step 1. 2. 3. 4. 5. 6.
Particle-free extract MnCl~ treatment Ammonium sulfate DEAE-cellulose Column electrophoresis c Gel filtration
Total units
Protein (mg/ml) b
Specific activity
Recovery (%)
1362 1309 765 515 333 293
17.8 9.9 40.1 12.6 10.2 11.7
0.0255 0.0306 0. 152 0.585 3.84 7.63
100 96 56 38 24 21
a Modified from J. Van Beeumen and J. De Ley, Eur. J. Biochem. 6~ 311 (1968). b Calculated from OD~80/OD~60, except for step 1, where protein is determined by the method of Lowry [O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 198, 265 (1951)]. c Results obtained with the Porath column, see text. cofactor of the enzyme. The difference spectrum shows a flavin peak at 455 nm, and a smaller one at 396.5, when the enzyme is reduced with lactose. Less pure enzyme preparations show the participation of cytochrome c in the oxidation of lactose. Good electron acceptors of the enzyme, apart from P~V[S and D I P , are KaFe(CN)6 and heart muscle cytochrome c. The oxidation rate of lactose is optimal at p H 7 with cytochrome c, at p H 6 with DIP, and at p H 8 with P M S as acceptor. Cyanide, azide, CO, E D T A , Ca 2+, and atabrine have no inhibitory effect on the enzyme. At 10 mM, cyanide does inhibit enzyme action b y 70%. Specificity. The first number after the name of each substrate mentioned below denotes its relative rate of oxidation. The next numbers are RI values of the corresponding 3-keto derivatives on cellulose thinlayer plates, developed in the solvent designated by letter in parentheses and identified under D e t e c t i o n of 3-Uloses. Migration values larger than 1 are calculated versus glucose; all others versus the solvent front. The data are lactobionate 100, 0.07 (A), 0.22 (B); cellobiose 100, 0.27 (A), 1.3 (A); methyl fl-D-glucose 85, 0.67 (A), 0.25 (E), 11.0 (E) ; methyl a-Dglucose 84, 0.67 (A), 0.51 (C), 11.0 ( E ) ; lactose 84, 0.06 (A), 0.23 (B) ; D-glucose 84, 0.60 (A), 0.49 (E), 19.0 ( E ) ; lactulose 76, 0.05 (A), 0.37 (B), 0.40 (D) ; p-arbutine 76; maltose 70, 0.20 (A) ; cellobionate 63, 0.26 (A); sucrose 60, 1.05 (A); D-galactose 56; glucose-l-phosphate 56; maltobionate 46, 0.28 (A) ; trehalose 30, 0.18 (A), 0.49 (B) ; fl-melibiose 25, 0.16 (A), 0.49 (B); 2-deoxy-n-glucose 22; leucrose 12, 1.10 (A); methyl-fl-n-thiogalactose 10, 0.45 (A), 0.67 (B), 0.37 (C); melezitose 10; D--mannose 10; raffinose 7, 0.09 (A); D-glucosamine 5; anhydro-l,6D-glucose 4; 2-deoxy-D-galactose 2. In terms of substrate configuration,
158
OXIDATION--REDUCTION ENZYMES
[36]
a sugar is oxidized by hexopyranoside: cytoehrome c oxidoreductase if it occurs in the hexopyranose C-1 chair form, has a hemiacetal oxygen or sulfur atom at C1, an equatorial OH group at Ca, and a C H 2 0 H group at C~. An equatorial configuration of the OH group at C2 and C4 is preferential over an axial one. There is no definite proof yet that the enzyme acts only on D-sugars. Km values measured with enzyme preparation from step 3 are cellobiose, 0.2 mM; lactobionate, 0.21 mM; maltobionate, 0.36 mM; lactose, 1.7 mM; maltose, 2.8 mM; glucose, 2.9 mM; sucrose 4.1 mM, and galacrose, 25 mM. D e t e c t i o n oJ 3-Uloses. These compounds can be detected either chromatographically on thin-layer plates, or polarographically as described elsewhere. 2 Solvents used for the chromatography are (A) acetone-acetic acid-water (20:6:5, v / v ) , (B) water-saturated phenol, (C) methylethylketone-acetone-water (30:10:6, v / v ) , (D) ethylacetate-pyridine-acetic acid-water (5:5:1:3, v / v ) , (E) water-saturated methylethylketone. T h e y display a specific gray-violet color at 100 ° with o-phenylenediamine spray reagent, 6, and a less sensitive reddish color with fluorescence after 10 min at 100 ° with an urea phosphate spray reagent. ~ The first reagent is made by suspending 400 mg of o-phenylenediamine in 3 ml of water, adding 0.65 ml of concentrated HCI, and diluting to 20 ml with ethanol, The second reagent contains 3 g of urea in n-butanol-ethanolwater-85% phosphoric acid (80:10:10:6, v / v ) . I t allows detection of 15 ~g of 3-ulose. The chemical structure of the 3-uloses of sucrose s,9 lactose ~,1° lactobionate, 6 maltose,6, ~o maltobionate, G trehalose, ~° cellobiose, 11 glucose, 12 methyl-fl-D-glucose, 1~ and glucose 1-phosphate ~4 has been established, and several of them have been crystallized 9,~. This is not yet the case for the other presumed 3-uloses. ' C. S. Wise, F. J. Dimler, H. A. Davis, and C. E. Rist, Anal. Chem. 27, 33 (1965). s M. J. Bernaerts, J. Furnelle, and J. De Ley; Biochim. Biophys. Acta 69, 322 (1963). 9S. Fukui, R. M. Hochster, R. Durbin, E. E. Grebner, and D. S. Feingold, Bull. Res. Counc. Isr. Sect. A 11, 262 (1963). 1oS. Fukui and R. M. l=Iochster, Can. J. Biochem. Physiol. 41, 2363 (1963). 11K. Hayano and S. Fukui, J. Biochem. 64, 901 (1968). 1~S. Fukui and R. M. Hochster, Y. Amer. Chem. Soc. 85, 1697 (1963). ~3B. Lindberg and O. Theander, Acta Chem. Scand. 8, 1870 (1954). ~S. Fukui, ]. Bacteriol. 97, 793 (1969).
A 3-KETO SUGAR-FORMING ENZYME
153
Properties'
Substrate Specificity. Of 55 compounds tested as possible substrates, the only six that are oxidized by this enzyme are those that possess the L-arabino configuration and their deoxy derivatives. These are L-arabinose, D-galactose, 6-deoxy-D-galactose (n-fucose), 2-deoxy-D-galactose, 3,6-dideoxy-D-galactose (abequose), and 6-iodo-6-deoxy-D-galactose. Compounds that do not serve as substrates at 33 mM concentrations are: D-glucose, L-glucose, D-mannose, L-mannose, D-altrose, D-allose, L-galacrose, L-fucose, L-rhamnose, 2-deoxy-D-glucose, 6-deoxy-D-glucose, 6-deoxy-D-allose, D-glucuronic acid, D-galacturonic acid, D-glucose 6-phosphate, D-galactose 6-phosphate, D-glucosamine, N-acetyl-D-glucosamine, methyl ~-D-glucoside, 2-acetamido-D-allose, 2-acetamido-n-altrose, D-fructose, L-fructose, L-sorbose, D-fructose 6-phosphate, D-xylose, L-xylose, D-lyxose, D-ribose, 2-deoxy-D-ribose, D-arabinose, D-xylulose, Da-glyceraldehyde, D-mannitol, D-glucitol, myo-inositol, L-arabitol, D-arabitol, xylitol, ribitoi, maltose, cellobiose, sucrose, lactose, melezitose, turanose, melibiose, trehalose, and raffinose. Nucleotide Specificity. Both NAD and NADP serve as coenzymes. Km values at pH 8.1 are 15 ~M for NAD + and 68 ~M for NADP +. E:qect of pH and Substrate Concentration on Reaction Velocity. At high substrate concentrations the reaction velocity is maximal at about pH 9.4 and about 40% maximal at pH 8.1. However, the affinity of the enzyme for its substrate is greater at the lower pH. The respective apparent Km values for D-fucose, D-galactose, and L-arabinose are 0.50, 0.17, and 0.14 mM at pH 8.1, and 6.6, 2.0, and 0.52 mM at pH 9.4. Stability. Sephadex G-200 fractions (in l0 mM sodium phosphate buffer, pH 7.0) are stable to freezing for at least 2 months.
[36] H e x o p y r a n o s i d e : C y t o c h r o m e c O x i d o r e d u c t a s e from Agrobacterium 1 By J. VAN BEEUMEN and J. DE LEY Itexopyranoside -~ acceptor --+ 3-keto hexopyranoside -{- reduced acceptor Assay Method
Principle. Enzyme activity can be followed either manometrically as oxygen consumption, using 5-methyl phenazinium methylsulfate (PMS) 1j. Van Beeumen and J. De Ley, Eur. J. Biochem. 6, 311"(1968).
154
OXID&TION--REDUCTION ENZYMES
[36]
as intermediate electron carrier, or spectrophotometrically, as decrease in optical density at 600 nm using 2,6-dichlorophenolindophenol (DIP) as terminal acceptor. The former assay is laborious but offers the possibility to analyze the reaction products. A brief description is given in another article. 2 The latter assay is quicker and is described below.
Reagents D I P , 0.885 m M in 0.4 M phosphate buffer, pH 6.0 Lactose, 0.1 M Water, distilled Enzyme, 0.002-0.05 unit/ml
Procedure. The reaction can be followed in any spectrophotometer at 600 nm. A cuvette of 1 cm light path receives 2.58 ml of D I P solution, 0.05-0.1 ml of enzyme, and water to a final volume of 2.7 ml. The amount of enzyme used depends on the possibilities of chart speed and scale expansion of the recorder. The reaction mixture is placed in the thermostated cuvette holder at 30 ° and, after 2 min of thermal equilibration, the endogenous reaction is recorded for 1 min. Enzyme activity is measured as the initial reduction rate of D I P after mixing in quickly 0.3 ml of lactose solution, kept at 30 °. When the endogenous activity is too high, a blank is included with 0.3 ml of water instead of the substrate. Since D I P is known to be a very good acceptor with many other enzymes, one should identify the 3-keto sugar (synonym: 3-ulose) formed, as described below, whenever the presence of a 3-ulose-forming enzyme is suspected in organisms other than Agrobacterium. Definition o] Unit. One unit of enzyme is the amount that oxidizes 1 ~mole of lactose per minute. Our sample of D I P having an extinction coefficient Cme = 11.85 cm -1 mmole -1 at pH 6.0, this amount of enzyme gives a decrease of 4 optical density units under the conditions of the assay. Specific activity is expressed as number of units per milligram of protein. Source o] Enzyme. The enzyme is inducible and is known to occur only in most strains of Agrobacterium tumefaciens and A. radiobacter. The enzyme was completely purified from cells of A. tume]aciens ATCC 143.1 Less pure preparations have been reported from A. tumefacien~ strain B6, 3 BNV6, a and IAM 1525. 4 The latter strain is derived from 2j. Van Beeumen and J. De Ley, this volume [3]. E. E. Grebner, E. Kovach, and D. S. Feingold, this series, Vol. 9 [17]. 4K. Hayano and S. Fukui, J. Biol. Chem. 242, 3665 (1967).
[36]
A 3-KETO SUGAR-FORMING ENZYME
155
strain ATCC 4452. The enzyme has also been called D-aldohexopyranoside dehydrogenase3,5 or D-glucoside 3-dehydrogenase2 Purification Procedure
Culture Conditions. We purified the enzyme starting from a 150-1iter culture, but the amounts can be scaled down if required. Agrobacterium tume]aciens ATCC 143 is grown for 40 hr at 30 ° in a culture containing 1% yeast extract, 0.1% KH2PO4, 0.2% (NH4)2S04, 0.025% MgSO~'7H20, and 1% lactose, final pH 6.8. Lactose is autoclaved separately in concentrated solution. Growth is started with a 5% inoculum. Good aeration (8 liters of air per hour liter of medium) and vigorous stirring are necessary. Approximately 2 kg of cells are harvested by centrifugation at the end of the logarithmic growth phase. They are washed three times with 10 mM phosphate buffer, pH 7.0. The following steps should be carried out at a temperature not exceeding 5 °. Step 1. Preparation of Particle-Free Extract. Portions of 35 g of washed cells in 70 ml of 10 mM phosphate buffer, pH 7.0, are disrupted in a Raytheon 10 kc sonic oscillator after replacement of the air in the cup by hydrogen. Unbroken cells and debris, removed by centrifugation, are resuspended, sonicated, and centrifuged again. The particles in the pooled supernatants are spun down for 1 hr at 100,000 g for 4 hr at 40,000 g. Step 2. Removal o] Nucleic Acids. To the particle-free supernatant, 0.05 volume of a 1 M MnCl., solution is added under stirring. After 30 rain the precipitate is discarded by centrifugation at 10,000 g. The supernatant is dialyzed against a 1000-fold excess of 10 mM phosphate buffer, pH 6.9. A heavy precipitate formed during dialysis is removed by centrifugation. Step 3. Ammonium Sul]ate Fractionation. To each liter of supernarant, 288 g of solid ammonium sulfate is added, and the precipitate formed is removed by centrifugation. The supernatant is then brought to 60% saturation by the addition of 61 g of solid ammonium sulfate to each liter. The precipitate is dissolved in 100 ml of 10 mM phosphate buffer, pH 6.9, and the residual salt is removed by three dialysis experiments against 5 liters of the same buffer. Step 4. DEAE-Cellulose Chromatography. The preceding preparation is chromatographed on a 4.1 X 68 cm column with 100 g of DEAE-cellulose (0.69 meq/g) equilibrated with 10 mM phosphate buffer, pH 6.9. E. E. Grebner and D. S. Feingold,Biochera. Biophys. Res. Commun. 19, 37 (1965). 6M J. Bernaerts and J. De Ley, J. Gen. Microbiol. 22, 137 (1960).
156
OXIDATION--REDUCTION ENZYMES
[36]
The ion exchange is pretreated in batch experiments with 0.2 N NaOH, washed with distilled water, charged with 0.1 M phosphate buffer, pH 6.9, until the effluent has the same pH, and equilibrated with the same buffer, diluted 10-fold. The enzyme is eluted under 1 meter of hydrostatic pressure in the 0.23-0.28 M KC1 fraction of a 3.4-liter linear gradient of 0 to 0.4 M KC1 in 10 mM phosphate buffer, pH 6.9. The enzyme-containing fractions are reduced in volume by precipitation with (NH4)2S04 and dialyzed as described at the end of step 3. Step 5. Column Electrophoresis on Agarose. The data for the electrophoretic purification step in the table were obtained after two subsequent zone electrophoretic runs on a large LKB 5801 Porath column.1 The following experiment on a smaller LKB 3340 column (3 }( 41 cm) gives the same purification factor and a slightly better yield (85%). The column is filled with 0.18% agarose in 33 mM phosphate buffer, pH 7.27. About 30 mg of protein in 3.5 ml is applied. Electrophoresis is continued for 38 hr at 250 V and 37-38 mA. Electrode buffer is renewed after 18 hr. At the end of the run, the gel column is lifted by hydrostatic pressure (15 cm) and collected in 2-ml fractions. Enzyme activity is measured in the presence of agarose. The agarose is centrifuged at 10,000 g for 20 min, rcsuspended in buffer and centrifuged again. The enzyme is prepared for step 6 by (NH~)~S04 precipitation and dialysis against 30 mM phosphate buffer, pH 7.17. Step 6. Gel Filtration. About 40 mg of protein in 4 ml of buffer are loaded on top of a 2.5 X 71 cm column of Sephadex G-75. Eluted at 25 ml/hour, the pure enzyme is collected as the first of two well separated protein peaks. The product is pooled, concentrated with (NH4)~S04, dialyzed against 50 mM phosphate buffer, pH 6.9, and stored under nitrogen at --18 ° when not in use. The purification procedure is summarized in the table.
Properties Stability. The enzyme becomes increasingly labile upon purification, perhaps owing to gradual loss of FAD. The activity half-life of pure enzyme is about a week when kept in the deep freezer. The enzyme is stable for 2 weeks under nitrogen at --15% The best pH range is 6.5-7.4. 20 % Physical and Chemical Properties. The sedimentation coefficient s0.75 of the oxidoreductase is 5.1 S. Its molecular weight is 85,000 __ 7700, as determined with Archibald's method, and assuming a partial specific volume ~ of 0.725. The molecular weight from gel filtration on Sephadex G-200 is about 15% smaller, probably because of a slight retention of the enzyme on the substratelike dextran matrix of the gel. FAD is the
[36]
157
A 3 - K E T O S U G A R - F O R M I N G ENZYME PURIFICATION OF HEXOPYRANOSIDE:CYTOCHROME C OXIDOREDUCTASE a
Step 1. 2. 3. 4. 5. 6.
Particle-free extract MnCl~ treatment Ammonium sulfate DEAE-cellulose Column electrophoresis c Gel filtration
Total units
Protein (mg/ml) b
Specific activity
Recovery (%)
1362 1309 765 515 333 293
17.8 9.9 40.1 12.6 10.2 11.7
0.0255 0.0306 0. 152 0.585 3.84 7.63
100 96 56 38 24 21
a Modified from J. Van Beeumen and J. De Ley, Eur. J. Biochem. 6~ 311 (1968). b Calculated from OD~80/OD~60, except for step 1, where protein is determined by the method of Lowry [O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 198, 265 (1951)]. c Results obtained with the Porath column, see text. cofactor of the enzyme. The difference spectrum shows a flavin peak at 455 nm, and a smaller one at 396.5, when the enzyme is reduced with lactose. Less pure enzyme preparations show the participation of cytochrome c in the oxidation of lactose. Good electron acceptors of the enzyme, apart from P~V[S and D I P , are KaFe(CN)6 and heart muscle cytochrome c. The oxidation rate of lactose is optimal at p H 7 with cytochrome c, at p H 6 with DIP, and at p H 8 with P M S as acceptor. Cyanide, azide, CO, E D T A , Ca 2+, and atabrine have no inhibitory effect on the enzyme. At 10 mM, cyanide does inhibit enzyme action b y 70%. Specificity. The first number after the name of each substrate mentioned below denotes its relative rate of oxidation. The next numbers are RI values of the corresponding 3-keto derivatives on cellulose thinlayer plates, developed in the solvent designated by letter in parentheses and identified under D e t e c t i o n of 3-Uloses. Migration values larger than 1 are calculated versus glucose; all others versus the solvent front. The data are lactobionate 100, 0.07 (A), 0.22 (B); cellobiose 100, 0.27 (A), 1.3 (A); methyl fl-D-glucose 85, 0.67 (A), 0.25 (E), 11.0 (E) ; methyl a-Dglucose 84, 0.67 (A), 0.51 (C), 11.0 ( E ) ; lactose 84, 0.06 (A), 0.23 (B) ; D-glucose 84, 0.60 (A), 0.49 (E), 19.0 ( E ) ; lactulose 76, 0.05 (A), 0.37 (B), 0.40 (D) ; p-arbutine 76; maltose 70, 0.20 (A) ; cellobionate 63, 0.26 (A); sucrose 60, 1.05 (A); D-galactose 56; glucose-l-phosphate 56; maltobionate 46, 0.28 (A) ; trehalose 30, 0.18 (A), 0.49 (B) ; fl-melibiose 25, 0.16 (A), 0.49 (B); 2-deoxy-n-glucose 22; leucrose 12, 1.10 (A); methyl-fl-n-thiogalactose 10, 0.45 (A), 0.67 (B), 0.37 (C); melezitose 10; D--mannose 10; raffinose 7, 0.09 (A); D-glucosamine 5; anhydro-l,6D-glucose 4; 2-deoxy-D-galactose 2. In terms of substrate configuration,
158
OXIDATION--REDUCTION ENZYMES
[36]
a sugar is oxidized by hexopyranoside: cytoehrome c oxidoreductase if it occurs in the hexopyranose C-1 chair form, has a hemiacetal oxygen or sulfur atom at C1, an equatorial OH group at Ca, and a C H 2 0 H group at C~. An equatorial configuration of the OH group at C2 and C4 is preferential over an axial one. There is no definite proof yet that the enzyme acts only on D-sugars. Km values measured with enzyme preparation from step 3 are cellobiose, 0.2 mM; lactobionate, 0.21 mM; maltobionate, 0.36 mM; lactose, 1.7 mM; maltose, 2.8 mM; glucose, 2.9 mM; sucrose 4.1 mM, and galacrose, 25 mM. D e t e c t i o n oJ 3-Uloses. These compounds can be detected either chromatographically on thin-layer plates, or polarographically as described elsewhere. 2 Solvents used for the chromatography are (A) acetone-acetic acid-water (20:6:5, v / v ) , (B) water-saturated phenol, (C) methylethylketone-acetone-water (30:10:6, v / v ) , (D) ethylacetate-pyridine-acetic acid-water (5:5:1:3, v / v ) , (E) water-saturated methylethylketone. T h e y display a specific gray-violet color at 100 ° with o-phenylenediamine spray reagent, 6, and a less sensitive reddish color with fluorescence after 10 min at 100 ° with an urea phosphate spray reagent. ~ The first reagent is made by suspending 400 mg of o-phenylenediamine in 3 ml of water, adding 0.65 ml of concentrated HCI, and diluting to 20 ml with ethanol, The second reagent contains 3 g of urea in n-butanol-ethanolwater-85% phosphoric acid (80:10:10:6, v / v ) . I t allows detection of 15 ~g of 3-ulose. The chemical structure of the 3-uloses of sucrose s,9 lactose ~,1° lactobionate, 6 maltose,6, ~o maltobionate, G trehalose, ~° cellobiose, 11 glucose, 12 methyl-fl-D-glucose, 1~ and glucose 1-phosphate ~4 has been established, and several of them have been crystallized 9,~. This is not yet the case for the other presumed 3-uloses. ' C. S. Wise, F. J. Dimler, H. A. Davis, and C. E. Rist, Anal. Chem. 27, 33 (1965). s M. J. Bernaerts, J. Furnelle, and J. De Ley; Biochim. Biophys. Acta 69, 322 (1963). 9S. Fukui, R. M. Hochster, R. Durbin, E. E. Grebner, and D. S. Feingold, Bull. Res. Counc. Isr. Sect. A 11, 262 (1963). 1oS. Fukui and R. M. l=Iochster, Can. J. Biochem. Physiol. 41, 2363 (1963). 11K. Hayano and S. Fukui, J. Biochem. 64, 901 (1968). 1~S. Fukui and R. M. Hochster, Y. Amer. Chem. Soc. 85, 1697 (1963). ~3B. Lindberg and O. Theander, Acta Chem. Scand. 8, 1870 (1954). ~S. Fukui, ]. Bacteriol. 97, 793 (1969).