[63] Glyoxylate dehydrogenase

342

D E H Y D R O G E N A SAND E S OXIDASES PURIFICATION OF

[63]

ENZYME

Fraction

Total protein (mg)

Total activity (units)

Units per rag protein

Per cent recovery

Extract Acid treated Alumina gel Sephadex G-200 TEAE-ceUulose Sephadex G-200

114,300 69,200 4,200 865 74 12

768 652 261 154 115 38

.0067 .0094 .0622 .178 1.56 3.16

100 85 34 20 15 5

has an absorption spectrum with maxima at 420, 320, and 278 m/~. This resembles the absorption spectrum of spinach ferredoxin. The activity of the enzyme is enhanced by the addition of either Clostridium or plant ferredoxin. When the enzyme is boiled, F M N in the supernatant fluid can be detected by chromatography. Enzyme activity is only slightly stimulated by addition of FMN. Physic.at Constants. The pH optimum is at 8.3. Since the enzyme is irreversibly destroyed in the absence of substrate, K~ values have not been determined. Inhibitors. The use of Tris buffer results in a 27% inhibition of activity at pH 8.3 as compared to phosphate buffer, p-Hydroxymercuribenzoate (10-4 M) immediately causes a 50-100% loss of activity which cannot be reversed with excess cysteine. The original form of the oxidase is inhibited only after an hour of incubation with 5 X 10-2 M PCMB. Fifty per cent inhibition of the enzyme occurs with about 3 X 10-~ M hydroxymethyl sulfonates or a-hydroxy-2-pyridylmethane sulfonate. The enzyme is not inhibited by sodium azide, sodium cyanide, or EDTA.

[ 63 ] G l y o x y l a t e D e h y d r o g e n a s e By J. R. QUAYLE Glyoxylate + CoA-SH + TPN + ~ oxalyl CoA + TPNH + H +

Glyoxylate dehydrogenase is an enzyme catalyzing the reversible oxidation and acylation of glyoxylate to oxalyl CoA, analogous to the acetaldehyde dehydrogenase discovered in Clostridiu~ kluyverii. 1 It has been found in oxalate-grown Pseudomonas oxalaticus and Pseudomonas IR. M. Burton and E. R. Stadtman, J. Biol. Chem. 202, 873 (1953).

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0DI, 2 and is probably present in shoots of Oxalis pes caprae2 In view of its probable metabolic function, viz. reduction of oxalyl CoA to glyoxylate, it may be termed oxalyl CoA reductase. The purification from Pseudomonas oxataticus and its properties which are described here have been published previously. 2,4

Assay Method

Principle. The enzyme is most conveniently assayed in the direction of glyoxylate oxidation, the equilibrium of the reaction being pulled toward oxalyl CoA formation by working at pH 8.6. Under these conditions, the assay is performed by measuring the rate of increase of optical density at 340 mt~ consequent on the reduction of T P N in the presence of glyoxylate and CoA. Cysteine is included in the reaction mixture to diminish oxidation of CoA; its presence enhances the linearity of the rate of T P N reduction at higher reaction rates. Reagents Sodium pyrophosphate buffer, 0.15 M, pH 8.6 Cysteine, 0.1 M Sodium glyoxylate, 0.1 M Coenzyme A, 0.01 M TPN, 0.01 M

Procedure. The complete reaction system in a 1.5-ml quartz cell (light path 1 em), consists of 0.2 ml of pyrophosphate buffer, 0.02 ml of cysteine, 0.05 ml of glyoxylate, 0.02 ml of CoA, 0.02 ml of TPN, enzyme extract, and water to total volume of 1 ml. CoA and T P N are omitted from the blank cell. The reaction is started by addition of TPN, whose reduction is followed at 340 m/~ over about 3 minutes. The initial rate is proportional to the amount of enzyme used over the range 0.002--0.02 unit. Definition o] Unit and Specific Activity. One unit of enzyme is defined as the amount that catalyzes the reduction of 1 micromole of T P N per minute under the conditions of assay. Specific activity is expressed as units of enzyme per milligram of protein. Growth Conditions. The source of Pseudomonas o$alaticus, its maintenance, and method of large-scale growth on oxalate is described elsewhere in this volume2 ~J. R. Quayle and G. A. Taylor, Biochem. J. 78, 611 (1961). ~A. Millerd, R. K. Morton, and J. R. E. Wells, Biochem. J. 88, 281 (1963). 4j. R. Quayle, Biochem. J. 87, 368 (1963). See this volume [67].

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DEHYDROGENASE8 AND OXIDASE$

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Purification Procedure

Step 1. Preparation o] Cell-Free Extract. Frozen bacteria (32 g wet weight) are crushed in a Hughes press at --25 ° and then mixed with 250 ml of 0.03 M phosphate buffer (pH 7.6) at 2 °. Crystalline ribonuclease and deoxyribonuclease (L. Light & Co., Ltd. Colnbrook, Bucks, England), 2 mg each, are added. The resulting extract is centrifuged at 16,000 g for 20 minutes and the precipitate is discarded. All subsequent operations are performed at 2 ° . Step ~. Treatment with Protamine Sulfate. Protamine sulfate is added to the extract in the proportion of one part to 10 parts of bacterial protein (w/w). The resulting suspension is centrifuged and the precipitate is discarded. Step 3. Ammonium Sul]ate Precipitation and Dialysis. Solid ammonium sulfate is added to the supernatant solution to give 40% of saturation. The precipitated protein is collected by centrifuging and discarded. To the supernatant solution is added ammonium sulfate to 60% of saturation. The pH of the solution is maintained at 7.0--7.2 throughout the precipitation procedure by addition of 0.01 N sodium hydroxide. The protein precipitating between 40 and 60% of ammonium sulfate saturation is collected by centrifugation and dissolved to a final volume of 7.6 ml in 0.03M sodium pyrophosphate buffer (pH 7.8), it is dialyzed for 1.5 hours against 3 liters of the same buffer. The resulting solution (11.7 ml) is stored overnight at --15 °. Step 4. Alumina C~f Gel Adsorption. The dialyzed fraction is diluted to 114 ml with water and the enzyme adsorbed on to C~,-gel by treatment with 0.34 volume of gel suspension (30.6 mg/ml). The gel is separated by centrifugation, and the adsorbed enzyme is eluted by four successive extractions with 30 ml of 0.01 M sodium phosphate-potassium phosphate buffer (pH 7.5). The four eluates are combined (113 ml). Step 5. Concentration o] Enzyme by Ammonium Sul]ate Precipitation, Followed by Dialysis. To the combined eluates solid ammonium sulfate is added to 80% of saturation. The precipitated protein is dissolved in 3 mM tris-HC1 buffer (pH 7.3) (final volume 9.6 ml) and dialyzed against 3 liters of the same buffer for 2 hours. Step 6. Ion Exchange Chromatography. A slurry of 5 g of diethylaminoethyl cellulose (DEAE-cellulose, Whatman DE 50) in 3 mM trisHC1 buffer (pH 7.3) is poured into a chromatographic column (2.5 cm >( 25 cm), and this is washed with 1 liter of tbe same buffer. The dialyzed extract is diluted with an equal volume of water and is applied to the column, which is subsequently washed with 200 ml of the same buffer. The enzyme is not eluted under these conditions. The column is then

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GLYOXYLATE DEHYDROGENASE

345

eluted with a solution of Tris-HCl buffer-potassium chloride in which the potassium chloride forms a linear gradient of increasing concentration. This is formed by drawing the column eluent from 600 ml of 3 m M Tris-HC1 buffer (pH 7.3) connected by siphon tube to 600 ml of molar potassium chloride. The levels of solution in both flasks should drop at the same rate throughout, and the flask containing the tris buffer should be stirred continuously. Fractions of eluate (approximately 6.5 ml) are collected at a flow rate of 40-50 ml per hour. Under these conditions the glyoxylate dehydrogenase is eluted mainly in five fractions around fraction number 50. These are combined, and the resulting solution (41 ml) is stored as separate 2-ml samples at --15 °. A summary of the purification procedure is given in the table. SUMMARY OF PURIFICATION PROCEDURE

Step

Volume Activity Protein (ml) (units/ml) (mg/ml)

1. Cell-free extract 210 2. Treatment with protamine 205 sulfate 3. 40-60% Ammonium sulfate 9.2 precipitate 4. Eluate from C~ gel adsorption 121 5. Dialyzed ammonium sulfate 9.6 precipitate 6. Pooled selected fractions 41 after column chromatography

1.87 1.93

6.4 6.3

Specific activity (units/rag)

Yield (%)

0.29 0.30

100 100

40.5

66

0.61

94

2.28 15.1

1.37 11

1.7 1.4

69 37

1.0

0.24

4.2~

10

a At the peak of activity eluted, the specific activity was 7.2.

Properties Specificity. As far as has been tested, the enzyme is specific for glyoxylate: no activity is observed with formaldehyde, acetaldehyde, glycolaldehyde, pyruvate, oxalacetate, a-ketoglutarate, or hydroxypyruvate. The enzyme is specific for T P N . CoA may be replaced by pantetheine, N-aeetylcysteamine reacts very slowly in place of CoA, cysteamine not at all. The preparation appears to be free of interfering enzymes. Activators and Inhibitors. No metal-ion requirements have been found. The enzyme is inhibited in the presence of Tris buffer. Replacement of pyrophosphate buffer in the standard assay system by Tris buffer of the same strength and p H halves the initial rate of the reaction. This fact, together with the increased hydrolysis of oxalyl thiol compounds which takes place in amino buffers such as Tris or triethanol-

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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 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,