serine and threonine deaminases of escherichia coli ... · serine and threonine deaminases of...

SERINE AND THREONINE DEAMINASES OF ESCHERICHIA COLI: ACTIVATORS FOR A CELL-FREE ENZYME*

BY W. A. WOOD AND I. c. GUNSALUS

(From the Laboratory of Bacteriology, Indiana University, Bloomington)

(Received for publication, June 17, 1949)

Serine has been reported by many workers to be deaminated by a variety of bacterial cells and tissues, but this process was first studied in detail by Gale and Stephenson in 1938 (1). These workers followed the serine deaminase of Escherichia coli by measuring the release of ammonia by resting cell suspensions. The reaction proceeded anaerobically, thereby distinguishing it from the oxidative deaminases. Aging of cell suspensions caused a loss of deaminase activity, which could be prevented by the addition of reducing agents, such as glutathione or formate, or by adenylic acid. Chargaff and Sprinson (2, 3) studied serine and threonine deaminases, using toluene-treated suspensions of E. coli and found that pyruvate and a-ketobutyrate, respectively, accumulated as the products of anaerobic deamination. Neither the O-ethers of serine nor phospho- serine were deaminated anaerobically. On the basis of these findings, Chargaff and Sprinson suggested desaturation as the mechanism. Binkley (4) obtained cell-free extracts of serine deaminase from E. coli which were inactivated by dialysis. The activity was restored by the addition of zinc ions. Lichstein et al. (5, 6) in studying the metabolic r61e of biotin inactivated the serine and threonine deaminases of E. coli by aging cell suspensions in phosphate buffer at pH 4. The activity was restored by the addition of biotin or adenylic acid. With a cell-free preparation, only yeast extract caused partial reactivation. From this evidence it was suggested that a coenzyme form of biotin is present in yeast extract (7).

In the present study, active serine and threonine deaminases have been obtained by growing E. coli in deep medium without carbohydrate. Vac- uum-dried cells were prepared which contained most of the deaminase activity present in the living cells, and which differed from the living cells only in the requirement of adenylic acid for activation. The deaminases were freed from the cells by autolysis, and purified by ammonium sulfate precipitation and adsorption on calcium phosphate gel. The purified enzyme required both adenylic acid (AMP) and glutathione (GSH) for activity. The enzyme, as prepared, deaminated both serine and threonine. During serine deamination, simultaneous inactivation toward both substrates occurred.

* This work KM : upportetl in part by the Office of Naval Research. 171

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172 HEHINE AND THREONINE DEAMKNASES

Methods Culture-The Crookes strain of E. coli, employed previously for studies

of the arginine and glutamic acid decarboxylases (8) and of tryptophanase (9), was used. For active deaminase production, the cells were grown without aeration in a medium composed of 2 per cent tryptone, 1 per cent yeast extract, and 0.5 per cent dipotassium phosphate. To obtain a large crop of cells, 10 liter batches were grown in 24 gallon reagent bottles. The medium was inoculated with 3 per cent of a 6 to 9 hour culture and incubated 12 to 14 hours at 37” (final pH 6.8 to 7.2). The cells were harvested with a Sharples centrifuge, the cell paste resuspended in 0.1 M

phosphate buffer, pH 7.8, containing 3 X lOAs M glutathione, and dried in vacua over Drierite (yield, 3.5 gm. of dry cells per 10 liters of medium). The deaminase activity of the dried cells was approximately 560 ~1. of pyruvate per mg. of dry weight per hour with n-serine and about 890 ~1. of a-ketobutyrate with L-threonine.

Determination of Serine and Threonine Deaminase-The deamination of serine and threonine has been shown to yield equimolar amounts of ammonia and pyruvate or a-ketobutyrate (3). Since the vacuum-dried cells did not metabolize these keto acids, the deaminase activity could be followed by measuring the rate of keto acid formation. The enzyme activity was assayed in a 1 ml. volume containing the following: 0.1 ml. of 1 M

phosphate buffer, pH 7.8, 0.1 ml. of 7 X lo+ M adenylic acid, water to 0.89 ml., 0.01 ml. of enzyme and 0.1 ml. (1 mg.) of n-serine or L-threonine. Before addition of the enzyme and substrate, the assay tubes were brought to 37”. After substrate addition, the reaction was allowed to proceed for 10 minutes, then stopped with 0.5 ml. of 20 per cent trichloroacetic acid, the protein removed by centrifugation, and a 1 ml. portion of the superna- tant removed for analysis.

A unit of serine or threonine deaminase was arbitrarily defined as the amount of enzyme necessary to form 1 PM of pyruvate or a-ketobutyrate in 10 minutes under the above experimental conditions.

Pyruvate-For most determinations, the direct method of Friedemann and Haugen (10) was used. Analyses by the extraction procedure of Friedemann and Haugen agreed with the results of the direct method.

ar-Ketobutyrate-a-Ketobutyrate was also determined by the direct method of Friedemann and Haugen. The color was compared with a standard curve prepared from crystalline a-ketobutyrate-2,4-dinitrophenylhydra- zone, m.p. 204-205” (uncorrected). The color, when read in the Evelyn calorimeter with a 515 rnp filter, was linear up to 70 y of a-ketobutyrate.

Results

The enzyme assay was standardized by using graded amounts of dried cells. As is shown in Fig. 1, nn-threonine was deaminated more rapidly


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W. A. WOOD AND I. C. GUNSALUS 173

than L-serine, the rate being proportional to cell concentration with each substrate. The formation of 20 to 120 y of pyruvate or 35 to 230 y of ar-ketobutyrate thus corresponds to 50 to 300 y of dry weight of cells. Since oxygen did not affect enzyme activity, assays were run aerobically. L-Threonine was used in later experiments with the purified enzyme and found to be deaminated more rapidly than the DL mixture, thus indicating - _ possible inhibition by the D isomer.

DEAMINASE- E.COLI 2 3.0

5

9

z

22.0

B 2

0

2 1.0

it xl

r a

0 100 200 300 400 500

E

pG DRIED CELLS PER ML FIG. 1. Serine and threonine deaminase activity of vacuum-dried cells. Condi-

tions, 0.1 ml. of M phosphate buffer, pH 7.8; 0.1 ml. (2.5 mg.) of adenosine-5-phosphate; 0.6 ml. of water; allowed to stand 5 minutes at 37”; cells as indicated and water to 0.9 ml.; 0.1 ml. (1 mg.) of L-serine or (2 mg.) nn-threonine; incubate 10 min- nutes at 37’; add 0.5 ml. of 20 per cent trichloroacetic acid. We wish to thank Dr. H. E. Carter for kindly furnishing the L-serine.

In the dried preparations, both deaminases required adenylic acid for activity, in contrast to the resting cells. In order to study the role of adenylic acid in the absence of interfering reactions, extraction of the enzymes and partial purification were undertaken.

Cell-Free Enzyme

4 gm. of vacuum-dried cells were suspended in 200 ml. of glutathione- phosphate buffer and the deaminases extracted by freezing, thawing, and autolysis. The cell-free extracts, obtained by centrifugation of the autol- ysate, contained about 50 per cent of the total activity. The enzymes


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174 SERINE AND THREONINE DEAMINASES

were precipitated by 40 per cent saturation with ammonium sulfate, ad- sorbed on calcium phosphate gel, and eluted with phosphate buffer. The details of the purification are shown in the flow sheet,. The first, eluate

Flow Sheet for Purification of Serine and Threonine Deaminases

Suspend 1 gm. vacuum-dried cells (4100 units serine deaminase, 4570 units threonine deaminase) in 50 ml. 0.1 M phosphate buffer, pH 7.8, containing 6 X 10-S M QSH. Freeze and thaw twice, autolyze 4 to 5 hrs. at 37”; centrifuge

I+--- 1 Discard 1870 units serine deaminase, 2420

units threonine deaminase Add (NH&SOI to 40yo saturation;

centrifuge I

II Redissolve in 5 ml. 0.1~ phosphat,e

buffer, pH 7.8, containing 3 X lo-’ M adenylic acid; centrifuge

I Discard

,L--p-- 1

Discard 1450 units serine deaminase, 1020 units threonine deaminase

Add 50 ml. 1 :lO dilution of Ca3(POJz gel, stir, centrifuge

lr- Wash with four 12 ml. portions

distilled water

-1 Discard

I----- - 1 Elute with 5 ml. M phosphate Discard washings

buffer, pH 7.8

---- 1

593 units serine deaminase, 525 units threonine deaminase; 10 to 15yo recovery

Add adenylic acid to 3 X 10es M

from the calcium phosphate gel contained 10 to 15 per cent, of the activity present in the dried cells. When stored in the frozen state, the enzymes were stable for several months; however, when stored at 0” without adenylic acid, inactivation occurred. The enzyme units recovered at, each step


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W. A. WOOD AND I. C. QUNSALUS 17s

in the purification and the degree of resolution with respect to adenylic acid (and glutathione) are shown in Table I. The degree of resolution was obtained by expressing the increase in rate of deamination due to addition of the activator as per cent of the maximum rate (activator present). The relative serine and threonine deaminase activity at each step in the purification is shown in the last column of Table I. The purity index was expressed as the ratio of threonine deaminase units to protein content, as determined by the biuret test of Robinson and Hogden (11).

Characteristics of Serine and Threonine Deaminases

The purified extract contained both serine and threonine deaminases in virtually the same proportions as were present in dried cells (Table I). This suggested a similarity of properties, if not the identity of the enzymes.

TABLE I Purijkation of Serine and Threonine Deaminases

Step No. Activity

1. Cell suspension (in GSH-0.1 M phosphate, pH 7.8)

2. Cell-free enzyme (from cells frozen, autolyaed at 37”, 5 hrs.)

3. Ppt. from 40% saturated (NH&zSOI 4. Eluate from Cas(PC& gel (in’1 M

phosphate, pH 7.8)

mits pm ccn; 4100 100

1870 46

1450 35 593 15

_-

Resolution

AhP per ccnl

82

94

GSH

___-

I.-SWiIlC ‘L-Th!O-

nine

0.90

0.77

1.42 1.13

For comparative purposes, the enzyme characteristics were investigated by use of both serine and threonine as substrates.

The influence of serine and threonine concentration upon enzyme activity is shown in Fig. 2. The half maximum activity was obtained with approximately 305 y per ml. of L-serine or L-threonine. This corresponds to a Michaelis constant (12) for substrate-enzyme of 3.5 X lo+’ and 3.0 X

1O-3 mole per liter, respectively. The presence of the D isomer of serine or threonine depressed deamination by about 50 per cent. Cysteine, which differs structurally from serine only in the polar group on the p-carbon, has been shown by Desnuelle and Fromageot (13) to be deaminated by E. coli in a manner similar to serine, the products being pyruvate, ammonia, and hydrogen sulfide. In the light of these similarities, desulfurase activity as determined by pyruvate formation was measured, but no activity was found. Binkley (4) has suggested that enolase, in addition to converting


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2lphosphoglyceric acid to phosphopyruvate, also catalyzes the deamination of cystcine and serine with the formation of pyruvate.

Adenylic Ac~G?-~~s with the dried cells, both serine and threonine cleaminases of the purified extract were activated by aclenylic acid. Aclenosine- Sphosphate obtained by hydrolysis of aclenosine triphosphate (ATP) or prepared by the yeast fermentation of aclenosinel gave the same activation (Table II). Other nucleosicles and nucleoticles including aclenosine, aclenosine-3-phosphate, and ATP were ineffective, thus indicating that adenosine-5-phosphate is specifically required. As is shown in Table II, yeast

$ 1.8 SUBSTRATE SATURATION

PURIYIED qEAMIN/ISES 1

-L-THREONINE

MILLIGRAMS SUBSTRATE PER ML

FIQ. 2. Substrate saturation curves for serine and threonine deaminases. Con- ditions as in Fig. 1 except as follows: 0.1 ml. of 1.3 X 10-l M glutathione added with adenosine-5-phosphate; allowed to stand 10 minutes at 37” with enzyme; substrate levels as indicated.

extract, which was found by Lichstein (7) to activate these deaminases in the absence of aclenylic acid or biotin, was ineffective with the purified enzymes.

The aclenylic acid activation curves for both cleaminases are shown in Fig. 3. The concentration necessary for half maximum activation is about 400 y per ml. with serine and about 245 y per ml. with threonine. These correspond to Michaelis constants of 1 X 1O-3 and 0.7 x 1O-s mole per liter respectively.

1 We wish to thank the Ernst Bischoff Company, Ivoryton, Connecticut, for a supply of adenosine-5-phosphate,


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W. A. WOOD AND I. C. OUNSALUS 177

Glutathione-For activity, the purified deaminase required glutathione in addition to adenylic acid (Table III). Reducing agents, including

TABLE II Activation of Serine and Threonine Deaminases by Adenosine-5-Phosphate

Additions

done .................................................. Adenosine ............................................. Adenosine-3-phosphate ................................ Adenosine&phosphate (from ATP) .................... Adenosine-5-phosphate (yeast fermentation). .......... Adenosine triphosphate. ............................... Guanylicacid ......................................... Yeastextract(lmg.perml.) ..........................

Additions ‘7 X 10-* M except as indicated.

T Keto acid formed

Pyruvate

Y

0

0

0

53 49

3.3 0 0

-__ ~-

r-Ketobutyrate

r

0

0

0

120 123

4.3 0 0

ADENOSINE- 5-PHOSPHATE SATURATION I PURIFIED DEAMINASE

oqyo L-SERINE

MILLIGRAMS ADENOSINE-5- PHOSPHATE PER ML.

FIG. 3. Adenosine-5-phosphate saturation curve for serine and threonine deaminases. Conditions as in Fig. 2 except 0.1 ml. (1 mg.) of n-threonine and adenylic acid levels as indicated. We are indebted to Dr. E. E. Howe of Merck and Company, Inc., Rahway, New Jersey, for a sample of n-threonine.

cysteine, sodium thioglycolate, and ascorbic acid, were ineffective. How- ever, sodium sulfide and sodium cyanide did cause partial reactivation. Since glutathione forms complexes with heavy metals and acts as a reduc-


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ing agent as well, other complex-forming agents were tested. Bipyridyl, 8-hydroxyquinoline, histidine, pyrophosphate, and gum arabic were with-

TABLE III Activation of PartiaElj/ Purified Serine and Threonine Deaminases

Conditions as in Fig. 3 except that glutathione and adenylic acid were added as indicated.

Additions Concentration

H x IQ-8

None ..................................... AMP.. .................................... 6.8 GSH. ...................................... 12.8 AMP + GSH.. ............................ 6.8, 12.8

- Keto acid formed

Pyruvate a-Ketobutyrate

7 7

0.5 0.0 2.3 3.6 6.1 4.9

107 134

THREONINE DEAMINASE GLUTATHIONE ACTIVATION

2.4 0 z

5 0 TIME WITH GSH IN MIN.

Fro. 4. Activation of threonine deaminaae by glutathione. Conditions as in Fig. 1 except as follows: Enzyme incubated with glutathione for the times indicated; adenylic acid added before or after incubation with glutathione as indicated; substrate, DL-threonine (2 mg. per ml.).

out effect, thereby suggesting the presence of oxidized functional sulfhy- dry1 groups rather than heavy metal inhibition. This possibility was


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further indicated by the fact that the enzyme is inhibited by lop5 M mer- curic, silver, and cupric ions, and is not reactivated by glutathione.

Enzyme activation by glutathione as a function of time is shown in Fig. 4. When glutathione was incubated with the enzyme in the presence of adenylic acid, activation was complete in 10 minutes. However, in the absence of adenylic acid partial activation occurred, followed by inactivation.

As with the dried cells, deaminase activity was proportional to the enzyme concentration. However, the rate of threonine deamination was

DEAMINASE ACTIVITY

WITH TIME 2.4

TIME IN MINUTES

FIG. 5. Serine and threonine deaminase activity as a function of incubation time Conditions as in Fig. 2 except for the time of incubation with substrate as indicated.

linear with time, whereas the rate of serine deamination decreased rapidly, approaching zero between 5 and 15 minutes (Fig. 5). The inactivation of the enzyme by serine occurred only during the reaction, that is, incubation of the enzyme in the presence of serine, but in the absence of the activators, or in the presence of the end-products, was without effect. Furthermore, increasing the concentration of each reactant did not pre- serve the rate of deamination. Similarly, the addition of yeast extract or a preparation of E. co& as a source of cofactors, neither preserved nor reactivated serine deaminase. The mechanism of the enzyme inactivation is not known.


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I THREONINE DEAMINASE-

INHIBITION BY SERINE I I I DL-THREONINE

8MG L-SERINE

0 5 IO 15 20 25 TIME IN MINUTES

FIQ. 6. Deaminase activity on mixtures of serine and threonine. Conditions as in Fig. 2 except for substrate mixtures and incubation times as indicated.

-. . I THREONINE DEAMINASE ;

’ I I INACTIVATION BY SERINE .‘,, -TYPCnh,,h,F

5 l.St---+-+- i”-

zl -/-

TIME IN MINUTES

FIQ. 7. Inactivation of threonine deaminase by serine. Conditions as in Fig. 2 except for substrate additions and incubation times as indicated.


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Deamination of serine and threonine by the purified extract may be due to the presence of two similar enzymes or one enzyme catalyzing the two reactions. As is shown in Table I, the purified preparation contained the deaminases in about the same proportion as the dried cells; also, as shown in Table III, both deaminases were activated by adenylic acid and glutathione. Attempts were therefore made to show the presence of separate enzymes catalyzing the two deaminations. The rate of keto acid formation in the presence of threonine with increasing levels of serine is shown in Fig. 6. The data indicate that the initial rate of deamination of a serine-threonine mixture was intermediate between the rates obtained with serine or threonine alone. Since the rates were not additive, competition for a single enzyme is suggested. A second fact which suggests the identity of the enzymes is the loss of deaminase activity for both substrates during incubation with the mixture, the rate being proportional to the serine concentration.

To show the presence of an independent threonine deaminase, conditions were employed in which serine deaminase was inactive; i.e., after 10 minutes incubation of the enzyme with n-serine (Fig. 5). The results, as recorded in Fig. 7, show that after incubation with serine the enzyme did not deaminate threonine, thereby indicating that threonine deaminase was incapable of functioning independently of serine deaminase. This suggests the identity of the two enzymes. Competitive inhibition of threonine deamination by serine appeared unlikely, since deamination of a serine- threonine mixture occurred at approximately the same rate as with threonine alone (Fig. 7).

SUMMARY

Serine and threonine deaminases have been obtained from Escher&h&x coli and partially purified.

The enzyme has been resolved and shown to require adenosined-phosphate and glutathione for activity.

Serine and threonine deaminases occurred in the extracts in approximately the same ratio as the dried cells, were activated by the same con- centrations of adenylic acid and glutathione, and threonine deamination disappeared when serine deaminase was inactivated. These facts suggest that both substrates may be activated by a single enzyme.

BIBLIOGRAPHY

1. Gale, E. F., and Stephenson, M., Biochem. J., 32, 392 (1938). 2. Chargaff, E., and Sprinson, D. B., J. Biol. Chem., 148,249 (1943). 3. Chargaff, E., and Sprinson, D. B., J. Biol. Chem., 161, 273 (1943). 4. Binkley, F., J. Biol. Chem., 160, 261 (1943). 5. Lichstein, H. C., and Umbreit, W. W., J. Biol. Chem., 170, 423 (1947).


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182 SERINE AND THREONINE DEAMINAYES

6. Lichstein, H. C., and Christman, J. F., J. Bid. Chem., 176, 649 (1943). 7. Lichstein, H. C., J. Biol. Chem., 177, 125 (1949). 8. Umbreit, W. W., and Gunsalus, I. C., J. Bid. Chem., 169, 333 (1945). 9. Wood, W. A., Gunsalus, I. C., and Umbreit, W. W., J. Biol. Chem., 170, 313

(1947). 10. Friedemann, T. E., and Haugen, G. E., J. BioZ. Chem., 147,415 (1943). 11. Robinson, H. W., and Hogden, C. G., J. Biol. Chem., 136, 707 (1940). 12. Lineweaver, H., and Burk, D., J. Am. Chem. Sot., 66, 668 (1934). 13. Desnuelle, P., and Fromageot, C., Enzymologia, 6, SO (1939).


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W. A. Wood and I. C. GunsalusENZYME

ACTIVATORS FOR A CELL-FREE COLI:DEAMINASES OF ESCHERICHIA

SERINE AND THREONINE

1949, 181:171-182.J. Biol. Chem.

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