/3-ASPARTOKINASE AND P-ASPARTYL PHOSPHATE

BY SIIMON RLACK AND NANCY G. WRIGHT

(From the Nationnl Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service,

Beth,esda, Maryland)

(Received for publication, July 26, 1954)

An enzymatic phosphorylation of L-aspartate, which yields P-aspartyl phosphate, was recently found to occur in yeast extracts (1). The reaction is as foll0ws:’

NHa+

I Mg++ ATP + -OOC-CH-CH,-COO- e

(1)

NHP’

I ADP + -OOC-CH-CH,-COPOa-

Subsequent work has shown that, in yeast extracts, P-aspartyl phosphate can be enzymatically reduced in two steps to form homoserine (2-5).

The enzyme which catalyzes Reaction 1 will be referred to as P-asparto- kinase. A description of some of its properties is presented here with a method for its preparation. A procedure for the chemical synthesis of p- L-aspartyl phosphate and some of its properties is also described.

EXPERIMENTAL

Preparation and Properties of P-L-Aspartyl Phosphate (BAP)

XynthesS-1.5 gm. (4.2 mmoles) of N-carbobenzoxy-L-aspartic acid a- benzyl ester, prepared according to Bergmann et al. (6),3 were used in each preparation. It was converted to N-carbobenzoxy-L-aspartyl oc-benzyl

1 Abbreviations used are ATI’ (ndenosinetriphosphate), ADP (adenosinediphos- phate), AMP (adenosine-5-phosphate), HAP (@-asparty phosphate), Tris (tris(hy- droxymethgl)mcth!I:lmine), TPSH (reduced triphosphopyridine nucleotide).

2 A brief description of this synthesis was previously published (5). 3 The melting point of carbobenzoxy-I>-aspartic acid anhydride, an intermediate

in this synthesis, was first reported by ihese workers to be 84” and later corrected to 124”. We found the 1, isomer to melt at 109-l 11”. This discrepancy has very recent11 been resolved by John and Young (7), who found that the compound can exist in two different crystalline states, one of which melts at 111” and one at 124”. A racemic compound was obtained by using acetic anhydride which analyzed substantially less than 97 per cent for the ring closure; this optically inactive preparation mrltrd at 127”.

27

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28 ASPARTOKINBSE AND ASPARTYL PHOSPHATP:

ester p-chloride by the procedure of the same authors. The latter com- pound was immediately suspended (partially dissolved) in 75 ml. of dry ether and transferred to a 250 ml. Erlenmeyer flask which contained an intimate mixture of 0.6 ml. of HsPOa (85 per cent) plus 1.2 gm. of AgsP04. The silver phosphate mixture, under the ether solution, was broken into small fragments with a stirring rod. A CaClz tube was then attached to the flask and the latter was mechanically shaken for 30 minutes at room temperature. The suspension was briefly centrifuged in capped tubes to give a clear ether solution of N-carbobenzoxy-L-aspartyl a-benzyl ester p-phosphate. The yield of this substance was about 80 per cent, as de- termined by the hydroxamic acid test for acyl phosphate. A qualitative test with AgN03, after heating a sample with HNOS, showed the absence of acyl chloride.

Removal of the carbobenzoxy and benzyl groups was achieved by cata- lytic reduction. The ether solution was transferred to a hydrogenation apparatus (Fig. 1) which was immersed in an ice bath and contained 15 ml. of water, 6.0 ml. of 2 M KHCO,, and 1 gm. of palladium black. Hy- drogen was passed through the apparatus at a rate sufficient to mix the ether and aqueous layers vigorously and keep the palladium suspended. This was continued until all of the hydroxamic acid-forming substance in the aqueous solution was reactive in the reverse reaction enzyme test de- scribed below. The ether by this time had completely evaporated. The solution was filtered by suction through the sintered disk of the hydrogen- ation vessel and, if necessary, separated from any toluene not carried away by the hydrogen stream. The pH was4 about 6.5.

The BAP concentration was 55 pmoles per ml., which indicates an over- all yield of 28 per cent. This solution was used in the experiments de- scribed here. It was stored at -2O”, where it deteriorated slowly but remained useful for several weeks. Attempts to purify the substance further as a silver, lithium, potassium, or barium salt, or on ion exchange columns, have invariably led to excessive destruction. It was possible, however, to remove most of the contaminating inorganic phosphate by slow addition of 1 M AgN03 while maintaining the pH at 6 to 7 with small additions of KOH.

Chromatographic Tests for Homogeneity of Synthetic BAP-Examination of the synthetic BAP by paper chromatography indicated the presence of only one ninhydrin-reactive substance, aspartic acid. Thus the acyl phos- phate was apparently split completely by drying its solution on t,he chro- matograph paper. The hydroxamic acid formed with hydroxylamine was also chromatographed on paper in a number of solvents and detected by

4 The pH was determirted at intervuls during the h~d~ogcfl:l,liorl. II’ il I’:r.lls I~!lo\\ 6.5, it should bo brought t,o 6..5 to 7.0 wiib sm:dl amounts of 5 N IiOlI,

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S. BLACK AND N. G. WRIGHT 29

the method of Stadtman and Barker (8). Only one hydroxamic acid was found. It behaved in all solvents exactly as synthetic P-aspartohydrox- amic acid. The hydroxamic acid derived from the enzymatically formed BAP also moved on paper chromatograms in this manner.

Chromatographic Differentiation of LY- and P-Aspartohydroxamic Acids- Separation of the CY and p isomers of aspartohydroxamic acid was achieved by qhromatographing them on Whatman paper No. 1 with a buffer-satu- rated phenol solution. The buffer used was 0.067 M nn-alanine adjusted to pH 10 with NaOH. By McFarren’s procedure (9) these substances were found to have average RF values of 0.20 and 0.26, respectively.

f H2 INLET

FIG. 1. Hydrogenation vessel

cY-Aspartohydroxamic acid was prepared by adding 0.1 ml. of 4 M hy- droxylamine hydrochloride to 5 mg. of isoasparagine and heating for 20 minutes at 100”. The product was placed on a small cation exchange column (the hydrogen form of Dowex 50, 50 mesh), washed with water, and the hydroxamic acid eluted with 0.5 M ammonium acetate buffer, pH 5.0.

fl-Aspartohydroxamic acid, when treated on chromatograph papers with ninhydrin, produces a brown color such as is formed by asparagine, the p-amide of aspartic acid. The a! isomers of the amide and hydroxamic acid produce a typical amino acid blue color.

Effect of pH and Temperature on BAP Stability-Fig. 2 shows the first order hydrolysis rates of BAP at three temperatures and several pH values. The curve representing rates at 30” may be compared to a very similar one reported by Koshland (10) for acetyl phosphate at 30”. The latter shows

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30 ASPARTOKINASE AND ASPARTYL PHOSPHATE

sharp upturns in hydrolysis rate at pH 1 and 11, compared to similar up- turns with RAP at pH 3 and 13. General stability of BAP is much lower, as its hydrolysis rate at pH 7 (30”) is 3 times the acetyl phosphate rate at this pH and 39”. Despite greater stability in alkaline solution in this experiment, storage at -20” is best at pH 7.

Use qf Lowry-Lopez Method with BAP-The relatively fast hydrolysis of BAP in weakly acid solution greatly limits the usefulness of the Lowry-

272tr_L11__1_1_I_J 175

150 3o”

72 x 100 s

D

Is”

50

0”

0 2 6 IO 14

FIG. 2. Hydrolysis rates of BAP. The final concentration of buffer was 0.09 molar in all cases, except at pH 13.5 (approximately), where 0.8 K KOH was used. Buffers employed were sodium sulfate, sodium formate, sodium phthalate, sodium acetate, sodium succinate, imidazole chloride, Tris chloride, ammonium chloride, and sodium carbonate. After the buffer solutions were equilibrated in the constant temperature baths, about 4rmoles of 13AP per ml. were added. BAP was det,ermined at zero time and at three subsequent times. This was done by pipetting 0.5 ml. of the buffered mixture into 0.5 ml. of 2 Y hydroxylamine (pH 8)) allowing it to stand 10 minutes, and then estimating the hydrosamic acid. The time intervals were chosen whenever possible to give approximately 30,50, and 70 per cent hydrolysis, but in no case exceeded a total of 6 hours. The rates were first order in all cases. The pH was determined at the end of each experiment with a Beckman model G pH meter with a glass elect rode.

Lopez method (11) for inorganic phosphate when BAP is present. This is especially true because small traces of certain proteins cause changes in the breakdown rate of BAP in the presence of the Lowry-Lopez reagents.6

* In an earlier expct,iment (1 ) acyl phosphate values obtained with the hydroxnmic acid mrthotl were in good agreement with acyl phosphal c vnlurs calculated from differences in inorganic phosphntr fouud with thr I”iske-Sul)L):Lro~~ (12) and Lowry- Lopez methods. This agreement was fortuitous, as we have since found both de- terminations for acyl phosphate, as used then, to give low recoveries with known amounts of synthetic HAP. Hydroxylamine at p1-I 4.0, used in the first experiment to stop enzyme activity as well as to determine BAP, forms only 60 per cent of the 0.aspartohydroxamic acid found when this reagent is used at pH 8.0.

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S. BLACK AND N. G. WRIGHT 31

Preparation of /3-Aspartokinase

Enzyme Tests-The activity of /3-aspartokinase may be determined by measuring the rate of either the forward or the reverse reaction. In the forward reaction test system, the following reagents, other than enzyme,

I .o

0 I 0 IO 20

MINUTES 0 0.1 0.2

ENZYME UNITS FIG. 3. A, Formation of hydroxamic acid in forward reaction enzyme test. 1 unit

of fl-aspartokinase a-as used. R, relation of enzyme concent,ration to the forward reaction rate; C, 13.41’ utilization in reverse reaction enzyme test (0.2 unit of enzyme was used); D, relation of reverse reaction rate to enzyme concentration. The ex- periments are described in the text.

are used in a final volume of 1 ml. All of these reagents, except MgC12, are brought to pH 8.0 with Tris base prior to addition: 100 pmoles of L-

aspartic acid, 20 pmoles of ATP, 20 pmoles of MgC12, and 400 pmoles of hydroxylamine hydrochloride. Hydroxylamine reacts with acyl phos- phate as it is formed to yield a hydroxamic acid. The latter is deter- mined after a 15 minute incubation at 15”. Crystalline fl-aspartohydrox- amic acid was used as a standard. 1 unit of enzyme causes the formation of 1 pmole of hydroxamic acid in this test. Fig. 3, A and R shows t)hat

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32 ASPARTOKINASE AND ASPARTYL PHOSPHATE

hydroxamic acid formation is proportional to incubation time and to en- zyme concentration.

In the reverse reaction test, final reagent concentrations in micromoles per ml. are 100 Tris chloride buffer (pH S.O), 10 ADP, 20 MgC12, and 2 to 4 BAP. The final volume is 0.5 ml. Following a 20 minute incubation at 15’, 0.1 ml. of 0.1 M sodium p-chloromercuribenzoate (a suspension) is added to stop the reaction. 5 minutes later 0.4 ml. of 2.0 M hydroxyl- amine is added. The hydrochloride of the latter is brought to pH 8.0 with Tris base prior to use. After 20 minutes, the hydroxamic acid is determined. Fig. 3, C and D shows that in this test utilization of BAP is proportional to incubation time and enzyme concentration.

/?-Aspartokinase activity can also be demonstrated in an optical test (Fig. 4). When this enzyme system is incubated together with aspartic

0 2 4 6 8 IO 12 MINUTES

FIG. 4. optical test for ~-Llsl)ariolti~l:tS(~. The complete system co~~(:ri~lcd in 1 ml.

t,he following, in micromoles: 100 imidazole chloride buffer (pI1 7.0), 0.081 Tl’KIl, 2 MgC12, 50 L- or n-aspartate, and 4 ATP. Also present were 0.8 unit of fl-asparto- kinase and 0.9 unit of aspartic p-semialdehyde dehydrogenase.

p-semialdehyde dehydrogenase (2) and TPNH, the BAP formed by the kinase is reduced by the dehydrogenase. TPNH disappearance, observed spectrophotometrically, is a measure of the reduction. The experiment shown illustrates the dependence of the test upon ATP and L-aspartate.

Yeast-6 pounds of fresh Anheuser-Busch bakers’ yeast were broken into fine particles by hand and quickly frozen by dropping into liquid Nz, con- tained in several large beakers.6 15 liters of Nz were required. The frozen yeast was stored in the beakers at -20” until used.

All of the following operations were carried out at O-5”. Extraction-250 gm. of the frozen yeast were mixed with 240 ml. of Hz0

and warmed to 0”. The pH was then brought to 8.3 to 8.5 with 5 M

NHJOH and the suspension stirred slowly for 2 days. About 300 ml. of extract were obtained by centrifugation at 12,000 X 9.

Acid and Protamine Treatment-The pH of the extract, now about 7, was

6 Loss of NS by evaporatjion was mir~imizcd by wrapping the beakers in paper.

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S. BLACK AND N. G. WRIGHT 33

adjusted to 5.1 with 2 M acetic acid and the inactive precipitate centrifuged. 15 ml. of 2 per cent protamine sulfate were added to the supernatant fluid and the precipitate was discarded after centrifugation.

Ammonium Suljate Precipitation and E&&on-The protamine super- natant fluid was mixed thoroughly with 25 gm. of Whatman cellulose powder (“standard grade”), and 125 ml. of saturated (0’) ammonium sul- fate were added slowly with stirring. The suspension was then poured into a 2.4 X 30 cm. glass tube such as is used for chromatograph columns. At the bottom was a coarse sintered glass disk and below this a stop-cock. The supernatant fluid was drained off and the column of precipitated pro- tein and cellulose washed with 25 ml. of 33 per cent saturated ammonium sulfate containing 0.02 M sodium acetate buffer, pH 5.0. The enzyme was

TABLE I

Summary of Enzyme Purification Data

The procedure is described in the text.

Extract ......................... Supernatant, pH 5.1.. ........... Protamine supernatant .......... 33% saturated (NH,) zSO, ppt.T. Eluate Fractions 25-27. .........

“ “ 28-30 ..........

300 240 252

18 18

7800 2260 4130 1610 2612 1600

287 1005 7.6 199

36 294

Units Yield, per mg. per cent

-___

0.29

0.39 71 0.61 71 3.50 44

26.2 9 8.2 13

* Data for the 33 per cent saturated (NH,) &Ol precipitate are based on a small scale pilot experiment.

then eluted by the procedure of Zahn and Stahl (13). A mixing chamber above the column contained initially 65 ml. of the 33 per cent saturated ammonium sulfate solution (buffered at pH 5.0). As this solution dripped onto the column, an equivalent volume of 28 per cent saturated ammonium sulfate-O.01 M KHC03 entered the mixing flask. The flow rate through the column was 30 ml. per hour. Effluent fractions of 6 ml. were collected. Most of the enzyme emerged as a sharp peak in twelve tubes centering about Fraction 28. This effluent was stored at -20’ and used in the experiments described here. Table I contains a summary of the purifica- tion data.

Properties of &Aspartolcinase Reaction

Metal Activation and E$ect of pH--@-Aspartokinase is activated by MC ions, which are maximally effective when the concentration is 0.03 M or more (Fig. 5). Mn+ ions are more effective at low concentrations, but

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34 ASPARTOKINASE AND ASPARTYL PHOSPHATE

reach a maximal activation lower than the Mg+ maximum. Fe++ ions are partially effective. No activation was found with Pb”, Ni*, Co*, Sn+, Ba++, Ca++, CL++, Zn++, or Fe+++.

Very little change in the reverse reaction is caused by varying the pH from 5.0 to 9.0. At pH 4.5, complete, irreversible inactivation occurred, and at pH 9.5 the rate was 60 per cent of that observed at pH 5 to 9. A

2.8

f \ 2.4

2 5 2.0 -I

5 I .6

8 0.4

f

MOLARITY OF DIVALENT ION FIG. 5. Activation of p-aspartokinase by- divalent metal ions. The conditions of

the reverse reaction test system were used in these experiments, except that the in- cubation time was 12 minutes. Each tube contained 0.16 unit of fi-aspartokinase. For each concentration of each metal a control was run without, ADP. BAP break- down due to metal addition was generally very small in the control tests. Mg++ and Mn++ were added as chlorides and Fe++ as Fe(KH4)2(S04)z.

quite similar relation of the forward reaction rate to pH has also been ob- served.

Synthesis of ATP in Reverse Reaction-Disappearance of BAP in the presence of ADP and enzyme is accompanied by a stoichiometric formation of ATP, as shown by the experiment recorded in Table II. These data verify the occurrence of the reverse reaction. In control experiments BAP did not disappear in the absence of ADP, and only a very small amount of ATP formed in the absence of BAP.

Equilibrium-The free reversibility of the reaction is illustrated in Fig.

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S. BLACK AND N. G. WRIGHT 35

6. With relatively high concentrations of L-aspartate and ATP, BAP accumulated until an equilibrium point was reached. Addition of ADP caused a fall in the BAP concentration $0 a new equilibrium level. If it is assumed that the appearance of ADP and the disappearance of ATP

TABLE 11 Enzymatic Formation of ATP from ADP Plus BAP

The complete system cont:Cned initially the following in micromoles per ml.: 50 of imidazole chloride buffer (pH 5.0), 10 of MgC12, 5 of ADP, 3.2 of BAP, and 0.8 unit of @-aspartokinnse. The reaction mixture was incubated for 15 minutes at 15”. The values are in micromoles per ml. of reaction mixture.

Complete system -ADP -BAP

Initial Final A Final Final _____--_

BAP. 3.20 0.62 2.58 3.15 ATI’............. 0.00 2.53 2.53 0.03

: 1.5 ADD ADP m

MINUTES

FIG. 6. Equilibrium of the p-aspartokinase reaction. The reaction mixture con- tained, initially, the following in micromoles per ml. : 250 of L-aspartate, 21.3 of ATP, 20 of MgCl,, and 1.6 units of p-aspartokinase. L-Aspartic acid was brought to pH 8.0 with Tris base prior to use. The incubation was at 15”. 2.86 pmoles of ADP were added in 0.06 ml. at the point indicated. BAP values subsequent to ADP ad- dition are corrected for this dilution. Values plotted are in micromoles per ml.

and aspartate are all equivalent to BAP formation, an equilibrium constant

K= BAP x ADP

L-aspartate X ATP

can be calculated from the data in Fig. 6. From several experiments such as the one illustrated, an average value of 3.5 X 1w4 has been obtained. This is very close to the value found for an analogous reaction involving ATP and 3-phosphoglyceric acid (14). It must be considered only an

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36 ASPARTOKINASE AND ASPARTYL PHOSPHATE

approximation of the true value, under these experimental conditions, be- cause side reactions have not been completely eliminated. The presence of some adenylokinase is indicated by the small formation of ATP from ADP in the absence of BAP (Column 6, Table II).

SpeciJicity-n-Aspartate, L-glutamate, and p-alanine are inactive as sub- stitutes for L-aspartate in the /3-aspartokinase reaction. AMP cannot sub- stitute for ADP (Fig. 3, C). In joint experiments with Dr. J. L. Strom- inger, uridine diphosphate, guanosine diphosphate, and inosine diphosphate were also found inactive as substitutes for ADP under the conditions de- scribed for the reverse reaction test.

DISCUSSION

Since BAP has not been isolated in a pure state or characterized on the basis of the usual criteria, the evidence for its nature is summarized here. The enzymatically formed substance, treated with hydroxylamine, yielded P-aspartohydroxamic acid which was identified chromatographically. It was quantitatively reduced, enzymatically, to L-aspartic fl-semialdehyde (2); the latter was identified and determined by a highly specific enzymatic method (3). A synthetic BAP has been made from a P-aspartyl chloride (with other reactive groups protected) by a method analogous to one used to prepare acetyl phosphate from acetyl chloride (15). The synthetic compound reacts in a manner similar to that of enzymatically formed BAP in the following tests. It yields P-aspartohydroxamic acid with hydroxyl- amine, it transfers phosphate to ADP in the presence of p-aspartokinase, and it is enzymatically reduced to L-aspartic /3-semialdehyde (2).

Subsequent papers (2-3) will describe how BAP is enzymatically reduced in two steps to homoserine, which is an intermediate in the biosynthesis of threonine and methionine (16). Genetic evidence (17) indicates that in Escherichia coli threonine and lysine have a common precursor which has not been identified. It is thus possible that BAP is involved, in some species, in the biosynthesis of lysine also. No evidence is available to support an earlier suggestion (1) that BAP is an intermediate in asparagine formation.

Materials and Methods

The sodium salt of ADP was prepared from the barium salt which was obtained from the Sigma Chemical Company. Disodium ATP was“ob- tained from the Pabst Brewing Company. TPNH was prepared by the procedure of Kaplan et al. (18). Ag,POJ was made by the method of Lip- mann and Tuttle (15). Crystalline P-aspartohydroxamic acid was pre- pared by a modification of the procedure of Roper and McIlwain (19). Isoasparagine was obtained from Dr. J. P. Greenstein.

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Simon Black and Nancy G. WrightPHOSPHATE

-ASPARTYLβ-ASPARTOKINASE AND β

1955, 213:27-38.J. Biol. Chem. 

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