[63] Guanylate kinase from Escherichia coli B

[63]

GUANYLATEK1NASE

473

weight of 21,300 are obtained by sedimentation analyses of the native enzyme. Sedimentation analysis and electrophoresis of the enzyme in the presence of strong denaturants show no evidence of subunit structure and yield molecular weight estimates of around 23,000.

Ultraviolet Absorbance. The enzyme demonstrates a broad absorbance maximum at 280 nm and 280:290 nm absorbance ratio of 1.84 at neutral pH. The absorbance coefficient is ~lcmPW~= 5.6 at 280 nm at neutral pH, which is similar to the enzyme prepared from rabbit muscle. 6

Catalytic Properties. Relative catalytic activity when tested using various nucleoside monophosphate and nucleoside triphosphate pairs shows: AMP + ATP (100%), AMP + dATP (52%), dAMP + ATP (10%), AMP + CTP (2.6%), CMP + ATP (1.1%), AMP + dGTP, GTP, UTP, and ITP (0.0%), and ATP + dGMP, GMP, UMP, and IMP (0.0%). Km values are 9 and 8 x 10-5 M for ATP and AMP, respectively, when tested at magnesium concentrations maintained at 2:1 ratio relative to ATP. A Km value of 1.1 X 10-4M is shown for ADP at constant 1.0 mM magnesium (at ADP concentrations tested at 0.010.05 mM). All other combinations of magnesium to ADP concentrations yield larger apparent Km values. Immunological Properties. Repeated injections of the enzyme in complete Freund's adjuvant into rabbit footpads at 3-week intervals yield no detectable antibody titer. The apparent lack of antigenicity is suggestive of a high degree of structural similarity between the human and rabbit enzymes. 6 L. Noda and S. A. Kuby, J. Biol. Chem. 226, 541 (1957).

[63] G u a n y l a t e

Kinase

f r o m E s c h e r i c h i a coli B

By MAx P. OESCHCER (d)GMP + (d)ATP ~ (d)GDP + (d)ADP

(1)

Guanylate kinase from E. coli actively phosphorylates guanine and 2aminopurine nucleofides but shows only slight activity with other pufine nucleotides and no activity with pyrimidine nucleofides. 1-a The enzyme 1M. J. Bessman and M. J. Van Bibber, Biochem, Biophys. Res. Commun. 1,101 0959). 2M. P, Oeschger and M. J. Bessman, J. Biol. Chem. 241, 5452 (1966). aE. G. Rogan and M. J. Bessman, J. Bacteriol. 103, 622 (1970). METHODS

IN ENZYMOLOGY,

VOL. LI

Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-5

474

PURINE METABOLIZINGENZYMES

[63]

will phosphorylate both ribo- and deoxyribonucleotide substrates although the Km value for dGMP is 5 times that of the Km value for GMP. z The activity of the enzyme is markedly stimulated by K + and NH4 ÷, but the mechanism o f activation by the two ions appears to be quite different. 2

Assay Methods Spectrophotometric Assay The spectrophotometric assay is the more convenient of the two assays presented but is subject to high blank values with crude extracts. Ninety percent of the interfering activities can be removed from crude extracts by treatment with acetone permitting use of the spectrophotometric assay at all stages of the purification. Principle. This method is based on the following sequence of reactions: (d)GMP + (d)ATP

guanylate kinase

2 phosphoenolpyruvate+ (d)ADP + (d)GDP

) (d)GDP + (d)ADP

(2)

pyruvate klnase

'

2 pyruvate + (d)ATP + (d)GTP

(3)

2 pyruvate + 2 NADH + 2 H+ lactat~Oehyd,o~n~e) 2 lactate + 2 NAD÷ (4) With pyruvate kinase and lactate dehydrogenase present in excess, the rate of guanylate phosphorylation is proportional to the rate of NADH oxidation, which is measured by the absorbance decrease at 340 nm. 4,5 Reagents. The composition of the assay mixture is shown in Table I. Procedure. The constituents of the assay (Table I) are mixed in a cuvette (1-cm path length), and the oxidation of NADH is followed spectrophotometrically at 340 nm. With impure enzyme preparations, a background rate of NADH oxidation is obtained for 2-3 min without (d)GMP in the reaction mixture. (Deoxy)guanosine monophosphate is then added to initiate the guanylate kinase catalyzed reaction. The background rate, which is due to ATPase and DPNH oxidase activities, is subtracted from the rate obtained after the addition of (d)GMP. Treatment of Crude Extract. The spectrophotometric assay can be used with crude extracts after removing proteins which give high 4A. Kornbergand W. E. Pricer, J. Biol. Chem. 193, 481 (1951). 51. Liberman,A. Kornberg, and E. S. Simms,J. Biol. Chem. 215, 429 (1955).

[63]

GUANYLATEKINASE

475

TABLE I SPECTROPHOTOMETR1C ASSAY MIXTURE Final

Volume

concentration

(ml)

(raM)

dGMP 0.1 M

0.39 0.10 0.12 0.20 0.05 0.02 0.04 0.03 0.02 0.03

-100 240 20 5 1 1 --3

Vf

1.00

Component H20 Tris.HC1, 1 M (pH 8 at 50 raM) KC1, 2 M MgCI2, 0.1 M ATP, 0.1 M (pH 7) Phosphoenolpyruvate, 50 mM NADH, 25 raM

Lactic dehydrogenase a Guanylate kinase ~

a Lactic dehydrogenase is Type I from Sigma which contains pyruvate kinase. Pyruvate

kinase is essential for the assay. The enzyme is prepared by dilution 1:5 with and subsequent dialysis against 0.05 M Tris'HC1 (pH 8 at 0.05 M) or 0.1 M KPO4 buffer (pH 7.5). b Highly purified samples of enzyme may be diluted in the solution of lactic acid

dehydrogenase to maintain full activity.

endogenous rates of N A D H oxidation in the absence of substrate. A convenient way to achieve this fractionation is to slowly add an equal volume of acetone (at - 1 0 ~) to a small volume of crude extract (approximately 10 mg of protein per milliliter) at 00. 6 The precipitate is removed by centrifugation and the acetone evaporated from the supernatant fluid by further centrifugation at 10° in an uncovered rotor. This treatment removes about 90% of the ATPase and DPNH oxidase activities from the crude extract while leaving the guanylate kinase activity unchanged?

Definition of Unit and Specific Activity. One unit of guanylate kinase activity is defined as the amount of enzyme which catalyzes the phosphorylation of 1.0/.~mole of (d)GMP per minute at 37 °. The specific activity of a preparation is defined as the units of guanylate kinase activity present per milligram of protein. 6The temperature may rise to 10° during the addition without affecting the guanylate kinase activity.

476

[63]

PURINE METABOLIZING ENZYMES

Radiochemical Assay Principle. In crude extracts the radiochemical assay gives the most accurate measure of enzyme activity. This method measures the formation of a phosphomonoesterase-insensitive material (nucleoside di- or triphosphate) from a phosphomonoesterase-sensitive material (nucleoside monophosphate). 7

Reagents. Composition of the assay mixture is shown in Table II. Procedure. The constituents of the assay (Table II) are mixed in a 13 x 100 mm tube and incubated at 37 ° for 20 min. s The reaction is TABLE II RADIOCHEMICALASSAY MIXTURE

Component

Volume (ml)

Final concentration (raM)

0.045 0.025 0.03 0.05 0.01 0.02 0.03 0.02 0.02

-100 240 20 5 4 -1 --

First incubation a

H20 Tris.HCl, 1 M (pH 8 at 50 raM) KCI, 2 M

MgCI~, 0.1 M ATP, 0.125 M (pH 7) Phosphoenolpyruvate, 50 mM Lactic dehydrogenase b [~P]dGMP, 12.5 mM (sp act -> 0.1 mCi/mM) Guanylate kinase Vf

0.25

Second incubation c

H20 Potassium acetate, I M (pH 5.1) Semen phosphomonoesterase

0.5 0.1 0.02

Norit adsorption a

HaPO~, 5 M Norit 20% (v/v) in 1-120

0.1 0.1

a Reaction set up on ice, incubated for 20 min at 37°, stopped by heating to 100° for 2 min. b Lactic dehydrogenase is Type I for Sigma which contains pyruvate kinase. The enzyme is prepared by dilution 1:5 with and subsequent dialysis against 0.1 M KPO, buffer, pH 7.5. e 30 min at 37°, stopped by placing tubes in ice-water bath. e Allowed to adsorb in ice-water bath for 5 min, Norit collected by filtration on RA 934 filters. 7I. R. Lehman, M. J. Bessman, E. S. Simms, and A. Kornberg, J. Biol. Chem. 233, 163 (1958). a The tubes are topped with glass marbles to prevent evaporation in this step.

[63]

GUANYLATE KINASE

477

terminated by heating the tube at 100 ° for 2 min. s The unreacted substrate is degraded following acidification of the reaction mixture with potassium acetate (pH 5.1) by incubation with a crude preparation of human semen phosphomonoesterase at 37 ° for 20 min. 9 After treatment with semen phosphomonoesterase the labeled product is adsorbed onto Norit, collected by filtration on a glass fiber disc, washed, dried, and counted. Purification

Procedure

2

The results of a typical purification procedure are summarized in Table III. After the third step (ammonium sulfate fractionation) the enzyme is relatively free of most noxious contaminants and can be used for the preparation of phosphorylated nucleotides. Unless otherwise stated all operations are carried out between 0 °--4 °. A buffer of 0.1 M potassium phosphate, pH 7.5, containing 1 mM EDTA is used. For large-scale work centrifugations are performed in a Lourdes continuousflow refrigerated centrifuge at 25,000 g. Step 1. Crude Extract. First 500 g of washed (0.5% NaC1, 0.5% KC1), packed E. coli B cells (stationary phase, grown aerobically at 37 ° in a medium composed of 1.1% K2HPO4, 0.85% KH~PO4, 0.6% Difco yeast extract, and 1% dextrose) are suspended in 5 liters of buffer with the aid of a Waring Blendor and then disrupted by sonic oscillation (a Branson sonifier tuned to maximum output fitted with a continuous-flow TABLE III PURIFICATION OF GUANYLATEKINASE FROM E. coli a

1. 2. 3. 4. 5. 6. 7.

Step and fraction n

Total volume (ml)

Crude (I) Acetone (II) (NI-~)2SO~ (IlI) K2HPO4 (IV) Sephadex (V) DEAE-cellulose (VI) DEAE-ceUulose (VII)

23,000 3,550 124 14 330 160 214

Specific Enzyme activity activity Protein (units/rag Relative Recovery (units/ml) (mg/ml) protein) purity (%)

0.22 1.0 24.8 200 6.4 8.4 4.3

8 3 26 99 0.43 0.22 0.023

0.027 0.33 0.95 2.0 15 38.2 187

1 12 35 74 556 1415 6926

100 70 61 56 42 27 18

a Activity was measured with the spectrophotometric assay using dGMP as the substrate. Steps 1, 2, 3, 5, and 6 were carried out on a smaller scale, and the fractions were stored until sufficient quantities were accumulated.

9j. Wittenberg and A. Kornberg, J. Biol. Chem. 202, 431 (1953).

478

PURINE METABOLIZING ENZYMES

[63]

attachment employing a flow rate of 18-22 ml/min). The supernatant fluid is collected by centrifugation 1° and is either stored frozen where it is stable or at 4 ° where it loses less than 10% of its activity per week.

Step 2. Acetone Fractionation. To each liter of fraction 1 (protein concentration 7-8 mg/ml) 1.5 liters of acetone are added over a 10-min period with constant, rapid stirring. 6 Immediately after the addition of the acetone the precipitate is removed by centrifugation at - 3 ° using a flow rate of 300 ml/min. 1° This supernatant fluid may be stored at - 1 5 ° for at least 1 week without the loss of activity. To each 1.5 liters of the supernatant fluid, at - 1 2 °, 0.8 liter of acetone at 0 ° is added with constant, rapid stirring over a period of 10 rain. During the addition the temperature is maintained at - 1 0 °. The precipitate is collected immediately by centrifugation at - 1 5 ° using a flow rate of 250 ml/min 1° and dissolved in buffer with the aid of a probe-type sonic oscillator. A ratio of 2.5 ml of buffer to 104 units of fraction 1 is used. Fraction 2 may be stored at 15° for at least 1 month with no loss of activity. -

Step 3. Ammonium Sulfate Fractionation. To 1.6 liters of fraction 2 (adjusted to 550 units/ml), 1.04 liters of saturated ammonium sulfate solution containing 1 mM EDTA are added with constant stirring over a period of 15 min. The suspension is allowed to equilibrate for 1 hr with constant stirring, and the precipitate is removed by centrifugation for 20 min at 16,000 g. To the supernatant fluid are added 925 ml of saturated ammonium sulfate solution, containing 1 mM EDTA, over a period of 10 min. The suspension is allowed to equilibrate for 1 hr with constant stirring, and the precipitate is then collected by centrifugation with a flow rate of 50 ml/min 1° and dissoh,ed in buffer (a ratio of 6 ml of buffer to 105 units of fraction 2). This fraction is stable for at least 6 months when stored at - 1 5 °. Step 4. Potassium Phosphate Fractionation. To 125 ml of fraction 3 (adjusted to 15,000 units/ml) 21 ml of 5 M K~HPO4 containing 1 mM EDTA are added drop by drop with constant stirring over a period of 5 min. The suspension is allowed to equilibrate for 10 min and the precipitate removed by centrifugation at 16,000 g for 20 min. To the supernatant fluid an additional 7.7 ml of the phosphate solution are added as above. After an additional 10 min for equilibration, the precipitate is collected by centrifugation for 20 min at 16,000 g and dissolved in 5 ml of distilled water. To this solution (13 ml) 1 ml of 1 M potassium phosphate buffer (pH 7.5 at 0.1 M) containing 10 mM EDTA is added. This fraction is stable at - 1 5 ° for at least 6 months. 1°For smaller volumes, batchwise centrifugation at 15,000 g for 20 min is used.

[63]

GUANYLATEKINASE

479

Step 5. Sephadex G-IO0 Fraction. A column of Sephadex G-100 (33.3 cm 2 × 100 cm) is equilibrated with 0.1 M potassium phosphate buffer, pH 7.5, containing 1 mM EDTA and 0. I M potassium chloride. Then 5 ml of fraction 4 are layered on the top of the column. 11 The sample is run into the gel, and the top of the column is layered with buffer and elution begun. The flow rate is set at 60 ml/hr, and 10-ml fractions are collected. The enzymic activity is quantitatively recovered in a peak between 0.39-0.46 column bed volumes. The peak fractions (with specific activities greater than 5000 units/mg protein) are pooled (fraction 5, Table III). The active fractions are conveniently identified because of the coincidental elution of a flavoprotein which is visible as a discrete yellow band migrating down the column. The pooled Sephadex fraction is stable for I week when stored at 4 °. Step 6. First Chromatography on DEAE-Cellulose. Fraction 5 (200 ml containing 85 mg of protein) is transferred by dialysis to 0.02 M potassium phosphate buffer, pH 7.0, containing 1 mM EDTA and applied to a column of DEAE-ceUulose (0.1 cm ~ × 100 cm) equilibrated with the same buffer. After washing the column with 15 ml of the phosphate solution, an exponential gradient is applied with 0 M and 0.2 M potassium chloride as limiting concentrations. The total volume of the gradient is 350 ml; 0,02 M potassium phosphate buffer, pH 7.0, and 1 mM EDTA are present throughout. The flow rate is set to approximately 7 ml/hr, and 3.5-ml fractions are collected. Of the activity applied, 80% is eluted in a single broad peak between 3.5-20 bed volumes (0.015-0.08 M potassium chloride). The peak fractions containing enzyme of specific activity above 20,000 units/mg of protein (64% of that applied to the column) are pooled (fraction 6, Table III). The flavoprotein which followed the enzymic activity in the previous column fractions is separated in this step. Analysis of the pooled fractions by disc electrophoresis reveals two major bands of protein. This fraction may be stored indefinitely at 4 ° without loss of activity. Step 7. Second Chromatography on DEAE-Cellulose. A column (0.1 cm 2 x 100 cm) is prepared with a washed DEAE-ceUulose suspension and equilibrated with 0.02 M potassium phosphate buffer, pH 8.3, containing 1 mM EDTA. Fraction 6 (135 ml containing 30 mg of protein) is equilibrated with the same buffer by dialysis and applied to the 11A convenient method for the even application of the sample to large diameter columns is to freeze it as a disc (2 mm thick, slightly smaller in diameter than the column i.d.) supported by a nichrome wire frame. The frozen disc is suspended just above the gel at the top of the column and allowed to melt. This method allows an even application of material without disturbing the surface of the gel.

480

PURINE METABOLIZING

[63]

ENZYMES

4000

50

I

.J

4o

CO

t-- 3 0 0 0 Z =)

"O X) 0

I->

30

rn

20

3 _

tO (l 2000 J,i (/) i . . . I.

Z

L.2

ILl < ._1 >Z

fO00

=)

0~ 0

5

0 IO

15

20

FRACTION NUMBER

FIG. 1. Chromatography of fraction 6 on DEAE-cellulose at pH 8.3. Activity was measured with the spectrophotometric assay using dGMP as substrate. The fractions indicated by the bar were pooled and constitute fraction7. FromM. P. Oeschgerand M. J. Bessman, J. Biol. Chem. 241, 5452 (1966). adsorbent at a rate of 12 ml/hr. The column is washed with 40 ml of the phosphate solution, and a linear gradient is applied with 0 M and 0.2 M potassium chloride as limiting concentrations. The total volume of the gradient is 1.5 liters, and 0.02 M potassium phosphate buffer, pH 8.3, and 1 mM EDTA are present throughout. The flow rate is set at 12 ml/ hr, and 22-ml fractions are collected. Of the activity applied to the column, 90% is eluted in a single peak between 2-60 bed volumes (0.003-0.08 M potassium chloride) (Fig. 1). The peak fractions (67% of the total activity) are pooled. As can be seen in Fig. 1 these fractions contain enzyme of constant specific activity (116,000 units/mg of protein) and reveal only one band when analyzed with disc electrophoresis. This fraction is stable indefinitely when stored at 4 ° in the eluting buffer or when dialyzed into 0.05 M Tris-chloride buffer at pH 8.1. Properties 2 Stability. The activity of guanylate kinase is stable at - 2 0 ° at all stages of the purification. The activity is unstable in dilute solution (less

[63]

GUANYLATEKINASE

481

than 5/xg protein per milliliter) but may be stabilized by the addition of bovine serum albumin or other suitable protein. Highly purified guanylate kinase is stabilized in the assay solutions by the presence of lactic dehydrogenase and pyruvate kinase.

Substrate Specificity. The activity of guanylate kinase is specific for purine nucleotides. It is highest with guanine, good with 2-aminopurine (one-tenth that of guanine), very limited with inosine (1% that of guanine), and barely detectable with adenine as the purine bases. The enzyme is active with ribo- or 2'-deoxyribonucleotides. The K m value for dGMP when using NH4 ÷ as the activating ion is 0.3 mM. Cationic Requirements. Guanylate kinase requires Mg 2÷ for activity. Mn 2+ will substitute for Mg z+ and to good measure so will Co 2+. The Km values for the three ions are 0.75 mM, 1.0 raM, and 1.25 raM, respectively. Guanylate kinase also requires either the monovalent ion K ÷ or NH4 + for activity. The apparent mechanism for activation by the two ions is quite different, for while both K ÷ and N H / enhance the Vmax, K ÷ reduces the Km value for dGMP 32-fold while NH4 ÷ does not alter the substrate Km value (Figs. 2 and 3). The Km values for K ÷ range between 10.8-347 mM while the Km value for NH4 ÷ remains fixed at 39 mM. 7 25 m M 6

35 m M

5

0

75 m M 4

I/v.

1~9 m M

3

2

)

-~2

.

-to

-e

-6

-4

-a

o

i

~

i

I

a

i

I

s

h

I

e

,o

I/dGMP (/j.. MOLES/ml) FIG. 2. Influence of K + on the Era for dGMP. Activity was measured with the spectrophotometric assay at five concentrations of K +. The values for the line marked were obtained by the extrapolation to infinite K + concentration of the observed rates with different concentrations of dGMP. The source of the enzyme was fraction 7. From M. P. Oeschger and M. J. Bessman, J. Biol. Chem, 241, 5452 (1966),

482

[63]

PURINE METABOLIZING ENZYMES 9 35 rnM

8

7 75 mM 6 189 mM 5

I/v. 4

524 m, 3

2 t

0 -4

-3

-2

-I

I+ I

I

I

I

I

2

3

4

5

I/dGMP (~.L MOLES/mr )

FIG. 3. Influence of NH4 + on the Km for dGMP. Activity was measured with the spectrophometric assay at four concentrations of NH4 +. The values for the line marked oo were obtained by the extrapolation to infinite N H + concentration of the observed rates with different concentrations of dGMP. From M. P. Oeschger and M. J. Bessman, J. Biol. Chem. 241, 5452 (1066).

pH Optimum. Guanylate kinase is maximally active in Tris-chloride buffer between pH 7.3 and pH 8.2. The enzyme shows 70% of its maximal activity at pH 6.5 and pH 9.0. Molecular Properties. Purified guanylate kinase migrates as a single sharp band during electrophoresis on 7.5% polyacrylamide gels. At equilibrium the purified enzyme distributes in a centrifugal field so that a plot of the log of the concentration versus the square of the radius of rotation gives a straight line. The slope of the line suggests a molecular weight for the native enzyme of 88,000. Fluorescence studies indicate that the enzyme may not contain tryptophan, for only a single emission maximum at 3.2 /~m is observed. These results indicate that the guanylate kinase activity is associated with a single protein, native molecular weight 88,000, which does not contain tryptophan.