[75] Glyoxylate reductase, two forms from Pseudomonas

[75]

GLYOXYLATEREDUCTASE, TWO FORMS FROM Pseudomonas

343

[CH3-(CH:)n-CO2H] are noncompetitive inhibitors with either glycolic or glyoxalic acid as the variable substrate and DCIP as the electron acceptor. Binding affinity increases as n is increased (e.g., Ki(,n~o)= 37 mM, Kl(n_5) = 92 pat/) and log K1 is a linear function of n. Dicarboxylic acids [HO~C-(CH2).-CO~H] are competitive inhibitors when glycolic acid is the variable substrate and DCIP is the electron acceptor but are noncompetitive inhibitors when glyoxalic acid is the variable substrate. Binding affinity decreases as n is increased (e.g., K i ( ~ o ) - 0.44 mM, K~¢nffi2)= 14 raM). Most inorganic anions, such as chloride and sulfate, are also inhibitors, but phosphate and arsenate cause enzyme activation, maximal at 0.1 M salt concentrations.1

[75] Glyoxylate Reductase, Two Forms from

Pseudomonas

1

By R. P. HULLIN Glyoxylate + NAD(P)H T H + -* glycolate ~ NAD(P) +

Two enzymes which catalyze the reduction of glyoxylate to glycolate may be obtained from Pseudomonas. Reduced pyridine nucleotides act as hydrogen donors for the enzymes. The NADH-linked glyoxylate reductase is entirely specific for its coenzyme, but the NADPH-linked reductase shows some affinity toward NADH.

Assay Method Principle. The enzymes are assayed by measuring the rate of oxidation of either NADH or NADPH in the presence of glyoxylate. The oxidation of the coenzyme is followed spectrophotometrically at 340 nm. Reagents Sodium glyoxylate, 1.0 M Phosphate buffer, 1.0 M, pH 6.8 NADH, 4 mM, in 1.0 M phosphate buffer, pH 6.8 NADPH, 4 raM, in 1.0 M phosphate buffer, pH 6.8

Procedure. The reaction mixture in 3.0 ml silica cells (l = 1) contains 0.3 ml of 1.0 M phosphate buffer pH 6.8, 0.1 ml of 1.0 M sodium glyoxylate, and an appropriate amount of enzyme. The reaction is started by the addition of 0.1 ml of 4 mM NADH or 4 mM NADPH, and the extinc1L. N. Cartwright and R. P. Hullin, Biochem. J. 101, 781 (1966).

344

OXIDATION--REDUCTION ENZYMES

[75]

tion at 340 nm is observed using a recording spectrophotometer at 25 °. At some stages, it is necessary to measure the rates of oxidation of N A D H and N A D P H in the absence of glyoxylate and use the values to determine the true enzyme activity. Activity. One unit of enzyme is defined as that quantity which gives AA34o,m = --0.001 unit/rain. Specific activity is defined as units of enzyme per milligram of soluble protein as determined by the optical density at 280 nm after correcting for nucleic acid absorption at 260 nm. ~

Purification Procedure The enzymes are obtained from a strain of Pseudomonas fluorescens isolated from soil which has the ability to grow on butane-2,3-diol as sole carbon source. Growth medium contains the following per liter: KH_oPO~, 5.0 g; (NH4)~SO~, 2.0 g; MgSO4.7H~O, 0.4 g; butane-2,3-diol, 2.0 g. The medium made up without the magnesium salt is adjusted to pH 7.0 with 5 M sodium hydroxide. After sterilization, the MgS04 is added aseptically as a 10% (w/v) solution. Cells from a fresh agar slope culture are used to inoculate two 50-ml samples of medium which after 24 hr of growth at 30 °, serve as inocula for 10 liters of medium in a 12-liter flask. After 24 hr of vigorous aeration, the crop of cells obtained is used to inoculate 40 liters of medium in a 55-liter aspirator containing 10 fishtank aerators, which is vigorously aerated at room temperature with a small amount of silicone MS antifoam to prevent excessive froth!ng. After 36 hr, the cells are brought into the logarithmic phase of growth by adding 5 liters of fresh medium and harvested at room temperature with a de Laval centrifugal separator. The cells, which have a distinct pink color en masse and wet weight of 1.0-1.5 g per liter of medium, are packed in stainless steel presses, 3 precooled to --14 °. After at least 6 hr at --14 °, the cells are crushed with the aid of a fly press. The crushed cells can be stored at --14 ° for up to 3 months without deterioration. Step 1. Extract. Frozen crushed cells (35 g) are thawed and diluted to 110 ml with 5 m M phosphate buffer, pH 7.0. Deoxyribonuclease (1 mg) and ribonuclease (1 rag) are added during the preparation of the homogeneous suspension. The extract is centrifuged at 70,000 g for 30 rain in a Beckman, Model L, preparative ultracentrifuge using a type 30 rotor at 2 ° and the precipitate is discarded. All subsequent operations are conducted at 2% 20. Warburg and W. Christian, Biochem. Z. 310, 384 (1941). 3D. E. Hughes, Brit. J. Exp. Pathol. 32, 97 (1951).

[75]

GLYOXYLATE REDUCTASE, TWO FORMS FROM

Pseudomonas

345

Step 2. Ultracentri]ugation. The supernatant solution is centrifuged at 140,000 g for 3 hr in a Beckman, Model L, preparative ultracentrifuge using a type 50 rotor and the ribosomal pellet is discarded. Step 3. Ammonium Sulfate. Solid ammonium sulfate is added to 30% saturation (17.6 g per 100 ml of extract), and the solution is continuously agitated. After allowing 20 min for equilibration, the precipitate is removed by centrifugation and discarded. More solid ammonium sulfate is added to the supernatant solution to 60% saturation (19.8 g per 100 ml of extract), and the previous procedure is repeated except that the precipitate is retained and dissolved in 30 ml of 5 mM phosphate buffer, pH 7.0. The solution obtained is dialyzed against 10 liters of 5 mM phosphate buffer, pH 7.0, which is replaced three times with 10 liters of fresh buffer at hourly intervals. Continuous agitation is essential to prevent excessive precipitation of the protein, and this is achieved by placing a 10-liter flask on a magnetic stirrer and allowing a plastlc-coated magnet to rotate inside the flask. Any small quantity of material which does precipitate during dialysis is removed by centrifuging. Step 4. Column Chromatography. DEAE-cellulose treated before use according to the method of Peterson and Sober ~ is suspended in 5 mM phosphate buffer, pH 7.0, and poured into a chromatography column (1.8 X 20 cm), the lower end of which is closed with a coarse sinteredglass disk. The column contents are equilibrated by allowing 1 liter of 5 mM phosphate buffer, pH 7.0, to run through it at 2% The dialyzed material is applied to the top of the column, at a rate just sufficient to keep the top moist; by this means the material can be adsorbed in a narrow band at the top of the colmnn; unadsorbed material is removed by passing 60 ml of 5 mM phosphate buffer, pH 7.0, through the column. A linear gradient of NaC1 is now applied to the column by allowing 200 ml of a solution containing 5 mM phosphate buffer, pH 7.0, to flow, with constant stirring, into 200 ml of a solution containing 5 mM phosphate buffer, pH 7.0, and 0.5 M NaC1; the resultant mixture is allowed to flow through the column at approximately 30 ml per hour, the effluent being collected continuously at 7.75 ml per fraction. The chloride content of the eluents is determined by the method of West and Coll.:' Two glyoxylate reductases are eluted from the column: (a) NADPHlinked enzyme is eluted between 40 and 100 mM NaC1, the peak of activity appearing at approximately 65 mM NaC1. Fractions 12-15 inclusive are used for further purification. (b) NADH-linked enzyme is eluted between 150 and 230 mM NaC1 with the peak appearing at 175 mM NaC1; fractions 23-26 are used for further purification. Those fractions contain4 E. A. Peterson and H. A. Sober, this series, Vol. 5, p. 3. ~P. W. West and H. Coll, J. Amer. Water Works Ass. 49, 1485 (1957).

346

OXIDATION--REDUCTION ENZYMES

[76]

ing each of the enzymes at a specific activity greater than in step 3 are combined, and the protein is precipitated with solid (NH4)2S04 to 60% saturation (39 g per 100 ml of solution).

Purification o] the NADPH-Lintced Glyoxylate Reductase Step 5. Gel Filtration, Sephadex G-200. The precipitate containing the NADPH-linked enzyme is dissolved in 1 ml of 0.1 M phosphate buffer, pH 7.0, is carefully placed on top of a column of Sephadex G-200, 55 X 2.25 cm, pretreated according to the method of Flodin 6 and equilibrated with 0.1 M phosphate buffer, pH 7.0. The bottom outlet of the column is then opened, and the enzyme sample is allowed to enter the bed surface; at the moment it disappears through the surface, a small amount of buffer is added to wash the surface. When this disappears a larger volume of buffer is added. The column is eluted by passing 300 ml of 0.1 M phosphate buffer, pH 7.0, at a rate of 5.5 ml per hour, the effluent being collected continuously at 2.75 ml per fraction. The enzyme is eluted between 115 and 154 ml of buffer collected and fractions 44-51 inclusive, which possess activity greater than 300 units/ml, are combined. Step 6. Precipitation with Ammonium Sul]ate. The pooled enzyme fractions are brought to 60% of saturation with solid (NH4)2S04 (39 g/100 ml.) After centrifugation, the precipitate is dissolved in 2 ml of water and diluted in the ratio 1 : 10 with buffer solution when required. A summary of the purification procedure, as presented in Table I, represents a 220-fold purification. Purification o] the NADH-Linked Glyoxylate Reductase Step 5. Gel Filtration, Sephadex G-200. The precipitate containing the NADH-linked enzyme is dissolved in 1 ml of 0.1 M phosphate buffer, pH 7.0, applied to the surface of a Sephadex G-200 column, 57 X 2.25 cm, and the column eluted with 300 ml of 0.1 M phosphate buffer, pH 7.0, as described in Step 5 of the purification of the NADPH-linked enzyme. The NADH-linked enzyme is eluted between 100 and 132 ml of buffer collected and fractions 4044 inclusive possessing activity greater than 3000 units/ml are combined. Step 6. Precipitation with Ammonium Sul]ate. The pooled fractions are brought to 60% of saturation with (NH4)2S04 (39 g/100 ml), and the enzyme precipitate, after centrifuging, is dissolved in 2 ml of water and diluted when required in the ratio 1:20 with buffer solution. 8p. Flodin, in "Dextran Gels and Their applications in Gel Filtration." Pharmacia, Uppsala, Sweden, 1962.

[75]

GLYOXYLATEREDUCTASE, TWO FORMS FROM Pseudomonas

347

TABLE I PURIFICATIONOF NADPH-LINKED GLYOXYLATEREDUCTASE

Step 1. Crude extract 2. Ultracentrifugation 3. (NH4)2SO4ppt. (30-60% sat.) 4. DEAE-cellulose 5. Gel filtration 6. (NH4)2SO4ppt. (60% sat.)

Volume (ml)

Total activity (units)

Total protein (mg)

94 84 38

46,800 43,700 39,600

1860 1250 538

27 21 2

22,400 13,400 12,200

32 2.7 2.2

Specific activity Recovery (units/rag) (%) 25.2 35.0 73.6 692 4920 6660

100 93 85 48 29 26

A s u m m a r y of the purification procedure is presented in Table I I ; it represents a 79-fold purification.

Properties 'Specificity and Kinetic Properties. The N A D H - l i n k e d enzyme is entirely specific for its coenzyme, but the N A D P H - l i n k e d reductase shows some affinity toward N A D H (relative activities toward N A D P H and N A D H in the ratio 4.4:1). Both enzymes convert h y d r o x y p y r u v a t e to glycerate. The preparation containing the N A D H - l i n k e d enzyme is contaminated with malate and lactate dehydrogenases; a gradual but not total removal of the contaminating enzymes is achieved during the purification procedure. The Km values for glyoxylate are 7 m M and 14 m M TABLE II PURIFICATIONOF NADH-LINKED GLYOXYLATEREDUCTASE

Step 1. Crude extract 2. Ultracentrifugation 3. (NH4)~SO4ppt. (30-60% sat.) 4. DEAE-ceUttlose 5. Gel filtration 6. (NH4)2SO4ppt. (60% sat.)

Volume (ml)

Total activity (units)

Total protein (mg)

94 84 38

231,800 219,300 176,800

1860 1250 538

28 13 2

103,500 61,800 58,200

90 7.8 5.9

Specific activity Recovery (units/mg) (%) 124.7 175.5 328 1150 7930 9870

100 95 76 45 27 25

348

OXIDATION--REDUCTION ENZYMES

[76]

for the N A D H - and NADPH-linked enzymes, respectively; for hydroxypyruvate, the corresponding K,,, values arc 5 mM and 7 mM. Maximal rate of glyoxylate reduction by both enzymes occurs with 33.3 inM sodium glyoxylate. Effect o] pH. Both reductases show maximal activity between pH 6.0 and 6.8, but the N A D P H - l i n k e d enzyme retains its activity over a wider range of pH values. Activators and Inhibitors. Oxo acids inhibit enzyme activity by combining with the active centers. Both enzymes are strongly dependant on free thiol groups for activity, as shown by inhibition with p-chloromercuribenzoate. The reduction of glyoxylate and hydroxypyruvate is not stimulated by anions. Physical Constants. The equilibrium constants for the reactions Glycolate + NAD + --~ glyoxylate + NADH + H + and Glycolate ÷ NADP + -~ glyoxylate -I- NADPH + H + are 6.0 X 10-is M and 3.0 X 10-is M, respectively. The molecular weights of the reductases are estimated by thin-layer gel filtration to be in the region of 180,000.

[76] A l d e h y d e D e h y d r o g e n a s e

from

Pseudomonas aeruginosa By W. E. RAZZELL Glycolaldehyde -b NAD +

K+

) glycolic acid -b NADH -t- H +

Assay Method 1

N A D H formation is determined in a silica cuvette containing the following components in a final volume of 1 ml: potassium phosphate, pH 7.2, 100 mM; 2-mercaptoethanol, 10 m M ; NAD ÷, 2 mM; enzyme. The reaction is started by addition of glycolaldehyde, 1 mM, after prior incubation for 5 min at 35 ° of all other components. Reaction is obtained with approximately 20 ~l of extract from cells grown on ethanol, or 1 ~g of purified enzyme. 1R. G, von Tigerstrom and W. E. Razzell, J. Biol. Chem. 243, 2691 (1968).