[64] Primary and secondary alcohol dehydrogenases from Gluconobacter

[64] Primary and secondary alcohol dehydrogenases from Gluconobacter

346 D~YD~OG~AS~S A~D GXIDAS~S [64] amine as compared with inorganic buffers such as phosphate, 6 makes Tris buffer unsuitable for use with the enzy...

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D~YD~OG~AS~S A~D GXIDAS~S

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amine as compared with inorganic buffers such as phosphate, 6 makes Tris buffer unsuitable for use with the enzyme. Equilibrium. Using a measured value for the equilibrium constant of glyoxylate dehydrogenase,4 the ~F' at pH 7 has been calculated to be --2.54 kcal. This compares with the value -b0.9 kcal calculated previously? Some of the free energy values used in the latter calculation are of doubtful accuracy. The equilibrium of the reaction may be upset if other thiol compounds besides CoA are present as a result of nonenzymatic ester interchange between oxalyl CoA and the thiol compounds. This effect becomes apparent at pH values above 7; at pH > 8 with excess cysteine present, the requirement for CoA becomes a catalytic one only. The reaction is readily reversible; at pH 6.5 in the presence of a fivefold excess of TPNH, oxalyl CoA may be reduced to glyoxylate within 1% of completion. ~ Stability. The activity of glyoxylate dehydrogenase rapidly diminishes in crude cell-free extracts, and losses of up to 70% have been observed within 1 day at 0 °. The enzyme becomes more stable on purification and may be stored at --15 ° for several weeks without loss of activity. Rather variable stability of the purified enzyme at 0 ° has been observed. pH Optima. The pH optimum for oxidation of glyoxylate in pyrophosphate buffer is 8.6. The optimum for reduction of oxalyl CoA to glyoxylate in phosphate buffer is 6.7. Kinetic Properties. The K~ values for glyoxylate and TPN, measured at pH 8.6 in pyrophosphate buffer at 25 °, are 5.7 X 10-4 M and 3.4 X 10-5 M, respectively. ej. Koch and L. Jaenicke, Ann. Chem. ~}5~ 129 (1962). TThe preparation and properties of oxalyl CoA have been described in the reference cited in footnote 4.

[64] P r i m a r y a n d S e c o n d a r y A l c o h o l D e h y d r o g e n a s e s f r o m Gluconobacter

By K. KERSTERS and J. DE LE¥ Acetic acid bacteria oxidize primary and secondary alcohols and glycols with a primary alcohol function to the corresponding carboxylic or hydroxyearboxylic acids. Secondary alcohols and glycols with a secondary alcohol function are oxidized to the corresponding ketoses. These oxidations are catalyzed by two different enzyme systems: one is soluble,

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the other particulate. 1,2 The particulate enzymes are probably localized on the cell envelope (considered to be the cytoplasmic membrane). This section describes the purification and the properties of two soluble NADlinked primary and secondary alcohol dehydrogenases and two particulate primary and secondary alcohol dehydrogenases from Gluconobacter oxydans (suboxydans), strain SU (NCIB 9108). I. S o l u b l e N A D - L i n k e d P r i m a r y a n d S e c o n d a r y Alcohol Dehydrogenases R--CH~0H

+ NAD

+ ~- R--CH0

+ NADH

R - - C H O H - - R ' + NAD + ~ R - - C 0 - - R '

+ H +

+ NADH + H ÷

Assay Method

Principle. The assay is based on the rate of reduction of NAD at 340 m~ in the presence of an alcohol. Reagents Tris(hydroxymethyl)aminomethane (Tris) buffer, 0.1 M, pH 8.8 MgC12, 0.05 M NAD, 0.O05 M Substrate: A 2 M solution is used for primary alcohols and glycols with a primary alcohol function. A 0.2 M solution is used for all other compounds. The primary and secondary alcohol dehydrogenases are routinely tested with respectively n-propanol and meso-2,3-butanediol as substrates. Enzyme. The enzyme is diluted in 0.01 M phosphate buffer at pH 7.0, to give a solution with an activity not greater than 0.3 unit/ ml.

Procedure. Six-tenths milliliter of Tris ml of NAD, and 0.1 ml of suitably diluted trophotometer cell with a 1-cm light path. the addition of 0.1 ml of alcohol solution. measured at 1-minute intervals.

buffer, 0.1 ml of MgCl_~, 0.1 enzyme are mixed in a speeThe reaction is initiated by The extinction at 340 m~ is

Definition oJ Unit and Specific Activity. One unit of alcohol dehydrogenase is defined as that amount which causes the reduction of 1 micromole of NAD per minute under the assay conditions described. Specific activity is expressed in terms of units of enzyme per milligram of protein, and protein is determined spectrophotometricaily. 3 ' K . Kersters and J. De Ley, Biochim. Biophys. Acla 71, 311 (1963). "~J. De Ley and K. Kersters, Bacteriol. Rcv. 28, 164 (1964). 3 O. Warburg and W. Christian, Biochem. Z. 310, 384 (1941).

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DEHYDROGENASES AND OXIDASES

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Purification Procedure All operations are performed at 0°-5 °, unless otherwise stated. Precipitates are separated by centrifugation at 15,000~ for 15 minutes, and dissolved in 0.01 M KH2PO4-Na2HP04 buffer at pH 6.5. Sodium chloride and ammonium sulfate are removed from solutions by gel filtration on Sephadex G-25 columns, equilibrated with the same buffer. Step 1. Growth of Culture and Preparation of Particle-Free Extract. Gluconobacter oxydans (suboxydans), strain SU, is grown at 30 ° on a solid medium containing 2% D-mannitol, 3% CaC03, 0.5% yeast extract, and 2.5% agar. The Roux flasks are inoculated with a 20-hour slant culture, grown on the same medium. The cells are harvested by centrifugation after 2 days of growth, and washed three times with 0.01 M phosphate buffer, pH 6.5. A cell-free extract is prepared by sonic disintegration of a 30% (w/v) suspension of the cells in 0.01 M phosphate buffer at pH 6.5 in a 10 kc, 250-watt Raytheon magnetostriction oscillator for 20 minutes at 4 ° under hydrogen atmosphere. Unbroken cells are removed by centrifugation at 20,000 g for 20 minutes. Centrifugation of the supernatant in a preparative Model L Spinco ultracentrifuge for 2 hours at 4 ° and 105,000 g results in the sedimentation of red particles, which also oxidize primary and secondary alcohols (see Part II). Step 2. Removal of Nucleic Acids. Two and half milliliters of 1.0 M MnC12 is added to 50 ml of supernatant solution. After 30 minutes, the precipitate is removed by centrifugation and discarded. MnC12 is eliminated from the resulting supernatant by gel filtration on Sephadex G-25. Step 3. Ammonium Sulfate Fractionation. Solid ammonium sulfate is added to 25% of saturation. The precipitate is removed by centrifugation and discarded. Ammonium sulfate is then added to 55% of saturation and the precipitate is collected by centrifugation and dissolved in 5 ml of 0.01 M phosphate buffer at pH 6.5. The ammonium sulfate is removed by gel filtration on a Sephadex G-25 column, previously equilibrated with the same buffer. Step 4. Chromatography on DEAE-Cellulose. The DEAE-cellulose column is prepared as described elsewhere in this volume. 4 The dialyzed ammonium sulfate fraction (7 ml) is applied to the column, and proteins are eluted with an increasing NaCl-gradient in the same buffer. The mixing chamber contains 450 ml of 0.01 M KH_~PO4-Na2HP04 buffer (pH 6.5), and the upper vessel contains 500 ml 0.15 M NaC1 in the same buffer. The flow rate is maintained at 40-50 ml per hour and fractions of 4 ml are collected. The primary and secondary alcohol dehydrogenases ' K. Kersters and J. De Ley, this volume [34].

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are eluted after 220 ml and 300 ml of buffer, respectively, passed through the column. Fractions 62-65, representing the peak of the primary alcohol dehydrogenase, and the fractions 84-87, representing the peak of the secondary alcohol dehydrogenase are collected and used to determine the enzyme specificity. A summary of the purification procedure is given in Table I. TABLE I PURIFICATION OF I~TAD-LINKED PRIMARY AND SECONDARY ALCOHOL ]~I~.HYDROGENASES

Primary alcohol Secondaryalcohol dehydrogenase dehydrogenase n-propanol meso-2,3-butanediol

Fraction Particle-free extract Ammonium sulfate (25-55%) DEAE-cellulose Fractions 62-65 Fractions 84-87

Specific Specific activity activity Volume Protein Total (units/mg Total (units/mg (ml) (mg/ml) units protein) units protein) 50 7 15 15

3.6 14 0.06 0.11

72 61 4.6 --

0.40 0.62

162 138

0.9 1.4

5.2 --

-18

-11.0

Properties The purified enzyme preparations are stable when kept frozen for at least 2 months. Both dehydrogenases show optimal activity between pH 8.8 and 9.2. Specificity. (1) NAD-LINXED PRIMARYALCOHOLDEHYDROGENASE.The enzyme is NAD-specific and catalyzes the oxidation of various primary alcohols and glycols with primary OH-groups at the following relative rates: n-propanol, 100; ethanol, 67; 1,7-heptanediol, 67; 1,6-hexanediol, 42; 1,5-pentanediol, 39; ethylene glycol, 28; 1,4-butanediol, 25; 1,3propanediol, 22; ethylene glycol monomethyl ether, 17; n-butanol, 17; n-hexanol, 16; allyl alcohol, 8; isobutanol, 3; and DL-1,3-butanediol, 2. The following compounds were found to be inactive: methanol, diethylene glycol, triethylene glycol, 2-ethyl-2-nitro-l,3-propanediol, sec-butanol, cyclopentanol, cyclohexanol, tert-butanol, DL-1,2-propanediol, meso-2,3butanediol, DL-2,3-butanediol, meso-3,4-hexanediol, glycerol, DL-lactate, and several polyols. (2) N A D - L I N K E D SECONDARY ALCOHOL DEHYDROGENASE. The enzyme is NAD-specific and catalyzes the oxidation of various secondary alcohols

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and glycols with a secondary OH-group at the following relative rates: DL-2,3-butanediol, 100; meso-2,3-butanediol, 50; (--)-3,4-hexanediol, 32; cyclooctanol, 15; cycloheptanol, 15; L-1,2-propanediol, 9; sec-butanol; 6; meso-3,4-hexanediol, 5; DL-1,2-propanediol, 5; cyclohexanol, 2; cyclopentanol, 1 and sec-propanol, 0.5. The following compounds were found to be inactive: primary alcohols and glycols with a primary OH-group, all polyols tested, glycerol, DL-1,3-butanediol, DL-lactate, DL-fl-hydroxybutyrate, and phosphoglycerate. II. P a r t i c u l a t e P r i m a r y a n d S e c o n d a r y Alcohol Dehydrogenases R--CH2OH --* R--CHO -b 2 H + q- 2 e R--CHOH--R' --~ R--CO--R' q- 2 H + ~ 2 e Assay Method

Principle. The particles contain the complete electron transport chain. The oxidation of alcohols and glycols by the particulate enzymes can thus be followed by measuring the rate of oxygen uptake in the Warburg respirometer. Once solubilized, the electron transport to oxygen is broken. Therefore the oxidation of primary and secondary alcohols is routinely determined spectrophotometrically by the reduction of 2,6-diehlorophenolindophenol. In spite of the disadvantages inherent to the use of this electron acceptor, the assay was found to be reproducible for routine determinations in following the purification of the enzymes. Reagent KH2PO4-Na:HPO~ buffer, 0.03 M, pH 5.8 2,6-Dichlorophenolindophenol, 10-3 M Primary or secondary alcohol, 0.2 M. Ethanol and meso-2,3-butanediol are routinely used as substrates. Enzyme. The enzyme is diluted in 0.03 M phosphate buffer at pH 5.8 to give a solution with an activity not greater than 0.2 unit/ml.

Procedure. One-tenth milliliter of suitably diluted enzyme, 2.9 ml of phosphate buffer, and 0.3 ml of 2,6-dichlorophenolindophenol are mixed in a colorimetric tube. The reaction is initiated by the addition of 0.2 ml of alcohol solution. The rate of decolorization is followed during 3 minutes at 660 m~ in a Beckman Model C colorimeter. The optical density change is calculated from the percentage transmittance recorded on a Varian recorder.

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Definition o] Unit and Specific Activity. One unit of primary and secondary alcohol dehydrogenase is defined as that amount which causes the oxidation of 1 micromole of alcohol, or the reduction of 1 micromole of 2,6-dichlorophenolindophenol per minute under the assay conditions described. In the Beckman C colorimeter, filter 66, with 12-mm tubes, 0.1 absorbance unit equals 0.06 micromole 2,6-dichlorophenolindophenol. Specific activity is expressed in terms of units of enzyme per milligram of protein, and protein is determined by the eolorimetric method of Lowry et al. 5 Purification Procedure All operations are performed at 0-5 ° unless otherwise stated. Precipitates are separated by centrifugation at 15,000 g for 15 minutes, and dissolved in 0.01 M K H 2 P 0 4 - N a 2 H P 0 4 buffer at p H 6.0. Sodium chloride, a m m o n i u m sulfate, and sucrose are removed from solutions by dialysis against 0.005 M phosphate buffer at p H 6.0. Step 1. Growth o] Culture and Preparation o] Particles. Cells of Gluconabacter oxydans (suboxydans), strain SU, are grown, harvested, washed, and sonicated as described for the soluble enzymes. The cell-free extract is centrifuged for 2 hours at 4 ° and 105,000 g in a preparative Model L Spinco ultracentrifuge. The red precipitate (particles) is suspended in 0.01 M KH2PO4-Na2HP04 buffer at pH 6.5 and again centrifuged at 105,000 g for 1 hour. Step 2. Solubilization o] Particulate Dehydrogenases. Washed particles (20 g, wet weight) are suspended in 60 ml 0.5% Triton-X-100 (Rohm & Haas Co, Philadelphia, Pennsylvania) in 0.025M KH2PO4Na2HP04 buffer with 10-4 M EDTA at pH 7.6. The mixture is stirred during 1 hour at 4 ° and centrifuged for 90 minutes at 105,000 g. Step 3. Removal o] Nucleic Acids. Three milliliters of 1.0 M MnCl~ is added to 60 ml of the red-pigmented supernatant solution. After 30 minutes a white precipitate is removed by centrifugation and discarded. MnC12 is eliminated from the supernatant solution by dialysis against phosphate buffer. Step 4. First Ammonium Sul/ate Fractionation. The dialyzed extract is fractionated with solid ammonium sulfate into the following fractions 0-30%, 30-40%, 40-45%, and 45-50%. The precipitate of each fraction is dissolved in 10 ml of 0.01 M phosphate buffer at pH 6.0 and dialyzed. Usually the 40-45% fraction, (NH4)~S0~-I, contains a tenfold purified 60. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

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ethanol dehydrogenase. It is, however, important to determine the activity in each fraction. The fraction with the highest specific activity possesses a pronounced red color. Step 5. Second Ammonium Sul/ate Fractionation. To the dialyzed 40-45% ammonium sulfate fraction, solid ammonium sulfate is added to 35% of saturation. The yellow precipitate is removed by centrifugation and discarded. Ammonium sulfate is then added to 80% of saturation, and the deep-red colored precipitate is dissolved in 3.5 ml of 0.01 M phosphate buffer (pH 6.0) and dialyzed [(NH4)~S04-2]. Step 6. Centri/ugation in a Sucrose Gradient. The sucrose gradients are prepared in 35 ml lusteroid tubes by layering sucrose solutions of 25.7%, 18.7%, 11.8%, and 4.9~ on top of each other. Linear sucrose gradients are obtained by leaving the tubes undisturbed overnight at 4 °. Eight-tenths milliliter of the (NH4)2S0~-2 fraction is layered on top of the gradient. The tubes are centrifuged in the Spinco SW-25 rotor during 8 hours at 25,000 rpm, the contents of the tubes are collected in several fractions. The peak of the alcohol dehydrogenase activity is located at approximately 12% sucrose. The fractions with the highest specific activity are pooled, dialyzed, and concentrated by saturating with ammonium sulfate. The red precipitate is dissolved in 3 ml 0.01 M phosphate buffer at pH 6.0 and dialyzed. A summary of the purification procedure is given in Table II. TABLE II PURIFICATION OF THE PARTICUI~ATE PRIMARY AND SECONDARY ALCOHOL I)EHYDROGENASES a

Fraction Washed particles Triton X-100 extract (NH4)~SO4-I (40-45%) (NHa)2S04-2 (35-80%) Sucrose-gradient centrifugation ~

Primary alcohol dehydrogenase ~

Secondary alcohol dehydrogenase~

Total units

Specific activity (units/mg protein)

750 52 34 8.4 5.4

0.40 0.08 0.46 0.48 1.5

Volume (ml)

Protein (mg/ml)

Total units

Specific activity (units/rag protein)

60 60 10 3.5 3

31 11 7.5 5 1.2

1210 935 540 156 84

0.65 1.4 7.2 9 24

a The activity of primary and secondary alcohol dehydrogenases is measured with, respectively, ethanol and meso-2,3-butanediol as substrates. b Represents ammonium sulfate precipitates of the most active fractions after sucrosegradient centrifugation.

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Properties No appreciable loss of activity occurred when the washed particles were kept frozen at --15 ° during 6 months. The purified enzyme is stable for at least 1 month under the same conditions. The optimal pH for oxidation of primary and secondary alcohols is 5.8 with a sharp decrease in activity below this point. Substrate Specificity. The purified enzyme has a very broad specificity. It catalyzes the oxidation of various primary and secondary alcohols, glycols, aldehydes, and polyols at the following relative rates. PRIMARY ALCOHOLS.Methanol, 10; ethanol, 100; n-propanol, 110; nbutanol, 110; n-amyl alcohol; 100; n-hexanol, 110; n-octyl alcohol, 90; isobutanol, 60; allyl alcohol, 100. GLYCOLS W I T H PRIMARY ALCOHOL FUNCTION. 1,2-Ethylene glycol, 30; 1,3-propanediol, 70; 1,4-butanediol, 80; 1,5-pentanediol, 80; 1,6-hexanediol, 90; 1,7-heptanediol, 140; DL-1,3-butanediol, 60; ethylene glycol monomethyl ether, 24; diethylene glycol, 1; triethylene glycol, 18; DL-1,2propanediol, 9; 1,2,6-hexanetriol, 16; 2-amino-2-ethyl-l,3-propanediol, 8; 2-nitro-2-ethyl-l,3-propanediol, 16. AROMATIC ALCOHOLS.Cinnamyl alcohol, 34; Anisyl alcohol, 32; coniferyl alcohol, 18; benzyl alcohol, 10. COMPOUNDS W I T H SECONDARY ALCOHOL FUNCTION. sec-Propanol, 32; cyclopentanol, 21; cyclooctanol, 14; cyclohexanol, 13; cycloheptanol, 11; 2,5-hexanediol, 11; meso-3,4-hexanediol, 9; meso-2,3-butanediol, 6; secbutanol, 5; DL-lactate, 4. Several polyols are only slowly oxidized. It was shown that the purified enzyme contained at least three different enzymes for the oxidation of alcoholic functions: (1) one or more primary alcohol dehydrogenase, (2) one or more secondary alcohol dehydrogenase, (3) one or more polyol dehydrogenase. The primary and secondary alcohol dehydrogenase are different enzymes, since 75% of the former and only 7% of the latter enzyme are solubilized by treatmerit with Triton X-100 (see Table II). Eighty percent of the meso2,3-butanediol dehydrogenase remains on the nonsolubilized fraction. The primary alcohol dehydrogenase is completely inhibited by 10-2 M p-chloromercuribenzoate, whereas the secondary alcohol dehydrogenase is not. The Triton X-100 treatment has thus released the enzymes from the bulk of the insoluble cell hull. Nevertheless we are led to believe that several apoenzymes are still linked together as a larger aggregate, as they could not be separated by column chromatography on several ion exchange resins (DEAE-, CM-, SE-cellulose, DEAE-Sephadex, and Amberlite XE-64). Electron Acceptor Specificity. For the crude particles oxygen could

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act as electron acceptor. In the purified state 2,6-dichlorophenolindophenol, ferricyanide, phenazine metbosulfate, thionine, and methylene blue are efficient electron aeceptors. Coenzyme. No evidence was found for the participation of NAD, NADP, FAD, FMN, the usual ubiquinones, and heavy-metal ions. All preparations display the spectrum of a reduced cytochrome-553 on addition of ethanol, meso-2,3-butanediol, or hydrosulfite. In all purification steps the ratio specific activity:specific hemoprotein content remained constant, indicating that enzyme activity is probably associated with this cytochrome-553. The purified enzyme in the oxidized form displays the following maxima: 530 m/~ (diffuse), 412 m/~, and 279 m~. In the reduced form the following maxima were observed: 553 m~, 522 m#, 418 m/~, 315 m/~, 279 n~, and a shoulder ~t 345 m/~. The spectral properties of this enzyme are identical with the ethanol-cytochrome-553 reductase from Acetobacter aceti (rancens).6 Inhibitors. The primary alcohol dehydrogenase is completely inhibited by 10-a M p-chloromercuribenzoate. The primary and secondary alcohol dehydrogenases are not inhibited by the following compounds (10-~M): arsenite, semicarbazide, hydroxylamine, cyanide, azide, ophenanthroline, 8-hydroxyquinoline, and EDTA. ST. Nakayama, J. Biochem. (Tokyo) 49, 240 (1961).

[ 65] E t h a n o l a m i n e O x i d a s e By STUARTA. NARRODand WILLIAM B. JAKOBY Ethanolamine + 1/2 02 --* glycolaldehyde + NH3 Assay Method 1 Principle. The assay is based on measuring the rate of formation of glycolaldehyde. This is accomplished by formation of the bis-2,4-dinitrophenylhydrazone of glycolaldehyde followed by alkali treatment and measurement of the resultant purple color. Reagents Sodium borate, 0.05 M, adjusted to pH 8.0 Ethanolamine, 0.025 M, adjusted to pH 8.0 with HC1 just prior to use. Commercial ethanolamine is redistilled and the fraction boiling between 171 ° and 173 ° is collected. IS. A. Narrod and W. B. Jakoby, J. Biol. Chem. 239, 2189 (1964).