activation of methionine for transmethylation” · activation of methionine for...

ACTIVATION OF METHIONINE FOR TRANSMETHYLATION”

II. THE METHIONINE-ACTIVATING ENZYME: STUDIES ON THE MECHANISM OF THE REACTION

BY G. L. CANTON1 AND J. DURELL (From the Laboratory of Cellular Pharmacology, National Institute of Mental Health,

United States Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, Maryland)

(Received for publication, September 13, 1956)

Vertebrates, higher plants, fungi, and presumably other organisms uti- lize the methyl group of methionine for biological methylations. As a result of recent work on the mechanism of transmethylation reactions, it has been established that, in reality, activation of methionine is a pre- requisite for the transfer of its methyl group (1, 2). The activation reaction is catalyzed by an enzyme system found in the livers of numerous mammalian species and in yeast. In this reaction, which is described by Equation 1, ATPI plays an essential role. More specifically, ATP fulfils a dual function, inasmuch as it serves (a) directly or indirectly as a source of the adenosine moiety that is incorporated in AMe (2) and (b) as an energy source, since it has been calculated (3,4) that the methylsulfonium bond in AMe is roughly equivalent to the pyrophosphate bond in ATP.

(1) GSH

n-Methionine + ATP A Mg++ S-adenosylmethionine + 31P

The mechanism of formation of AMe from methionine and ATP poses a number of interesting problems. As described by Equation 1, in the course of the activation reaction all three phosphates of ATP appear as IP. This can be explained only by assuming that the reaction mechanism

* Seventh paper in a series on enzymatic mechanisms and transmethylation. Preliminary reports were presented before the Third International Congress of Bio- chemistry at Brussels, 1955, and before the meeting of the American Society of Bio- logical Chemists at Atlantic City, April, 1956. Some of the experiments reported were performed by one of us (G. L. C.) while he was at the Department of Pharma- cology, School of Medicine, Western Reserve University, Cleveland, Ohio.

1 The following abbreviations are used throughout: ATP (or ARP-P-P) adenosine triphosphate; ADP, adenosine diphosphate; AMP, 5-adenylic acid; UTP, uridine triphosphate; ITP, inosine triphosphate; AMe, S-adenosylmethionine; IP, orthophosphate; PP, inorganic pyrophosphate; GSH, reduced glutathione; Tris, tris(hy- droxymethyl)aminomethane; MAE, methionine-activating enzyme of rabbit liver; YPPase, highly purified yeast inorganic pyrophosphatase; ATPase, adenosinetri- phosphatase activity of rabbit liver; LPPase, inorganic pyrophosphatase activity of rabbit liver.

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1034 ACTIVATION OF METHIONINE

is complex and that secondary hydrolytic steps are involved. Another point of interest relates to the manner in which the energy of the pyrophosphate bond of ATP is utilized to generate a new type of energy-rich bond.

The present communication deals mainly with the possible mechanism of the enzymatic activation reaction catalyzed by a partially purified preparation of MAE from rabbit liver and, more specifically, with the fate of the individual phosphate groups of ATP as investigated with isotopically labeled compounds.

EXPERIMENTAL

Methods

Enzyme Preparations; Methionine-Activating Enzyme-Rabbits of medium size and,of various breeds, most frequently albino, were used. Two rabbits can be used most conveniently for one preparation. The animals were anesthetized (usually 15 to 20 hours after the last feeding) by intra- venous injection of a 10 per cent solution of sodium Seconal (0.4 to 0.5 ml. per kilo of body weight) and bled from the carotids. The livers were then removed and placed on ice. All subsequent operations, unless other- wise stated, were carried out in a cold room maintained at 2’; glass-dis- tilled water was used throughout.

Step 1. Extraction and Ammonium Sulfate Fractionation-The livers were homogenized in a Waring blendor with 2.5 volumes of cold 0.01 N acetic acid. The suspension was centrifuged for 30 minutes in a Servall centrifuge at 18,000 X g. The supernatant fluid was decanted through a funnel containing a small glass wool plug and cooled to 2-4”. (The temperature of the suspension may rise to 12-15” during centrifugation.) Next a solution of ammonium sulfate saturated at 2” was added slowly with sufficient stirring (50 ml. per 100 ml. of solution). The mixture was stirred gently for 20 minutes, and then centrifuged in a Servall centrifuge (10 minutes at 18,000 X g). The precipitate was discarded, and additional ammonium sulfate solution was added to the supernatant fluid (36 ml. for each 100 ml. of initial volume). After an interval of 20 minutes, the precipitate was collected by centrifugation, the supernatant fluid was discarded, and the tubes were allowed to drain for a few minutes in the cold room. When stored at -2O”, the ammonium sulfate paste (Ammonium Sulfate Precipi- tate I) is stable for several weeks.

Step 2. Dialysis and Isoelectric Precipitation-The ammonium sulfate paste was dissolved in a small volume (10 to 20 ml.) of ice-cold potassium phosphate buffer (0.1 M, pH 6.3), and dialyzed for 4 hours against 0.05 M

buffer at the same pH in a rocking dialyzer. At the end of the dialysis, a small precipitate was removed by centrifugation, and the protein con-


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G. L. CANTON1 AND J. DURELL 1035

centration of the supernatant fluid was adjusted to 8 to 10 mg. per ml. by dilution with water. Protein was determined by the spectrophotometric method of Warburg and Christian (5). After addition of neutralized GSH (2 mg. per ml. of protein solution), the pH was lowered to 5.1 by the addition of approximately 0.01 volume of 1.0 M sodium acetate buffer, pH 4.9. The suspension was kept at 0” for 15 minutes and then centrifuged in the International refrigerated centrifuge. The supernatant fluid was discarded, and the precipitate was dissolved in approximately four-fifths the initial volume with use of 0.05 M phosphate buffer, pH 7.0, containing 2.0 mg. of GSH per ml. (Isoelectric Precipitate). The resulting solution could be stored frozen for several days with little loss of activity.

Step 3. Ammonium Sulfate Fractionation--The solution obtained above was diluted with water to a protein concentration of approximately 10 mg. per ml., and the pH was adjusted to 6.0 by addition of sodium acetate buffer (1.0 M, pH 5.5): The suspension was kept in an ice bath for 15 minutes and then stirred gently at room temperature until the temperature rose to 15-18” (usually 45 to 60 minutes were required for this step). After the suspension was cooled to 2”, a fairly copious precipitate of inert protein was removed by centrifugation, and the supernatant fluid was fractionated by the addition of saturated ammonium sulfate adjusted to pH 6.1 and containing Versene (1 mg. per ml.). The fraction precipitating between 33 and 46 per cent saturation contained most of the activity. The precipitate obtained by the above procedure was dissolved in 5 to 10 ml. of 0.05 M Tris buffer, pH 7.5, with 2 mg. of GSH per ml. (Ammonium Sulfate Precipitate II).

Step 4. Heat Treatment and Repreci@ation with Ammonium Sulfate- The enzyme solution was dialyzed for 4.5 hours against 500 volumes of 0.05 M phosphate buffer, pH 6.3. Next it was mixed with 2 volumes of a solution of the following composition: ATP, 0.02 M; L-methionine, 0.02 M; Tris buffer, 0.1 M, pH 7.5; MgCL, 0.3 M; and GSH, 0.008 M. The solution was quickly brought to 54-55” and kept at this temperature for 8 minutes. After being cooled to 2” in an ice bath, 2 volumes of saturated ammonium sulfate solution were added, and the suspension was centrifuged at high speed. The precipitate was suspended as well as possible in a solution containing 4 parts of saturated ammonium sulfate and 2 parts of neutralized GSH (12.5 mg. per ml.) and was recentrifuged. The supernatant fluid was discarded and the precipitate dissolved in 5 to 10 ml. of 0.05 M Tris buffer, pH 7.4, with 2.0 mg. of GSH per ml. At this stage the enzyme is stable for several weeks if kept frozen.

The fractionation described above results in 15- to 30-fold purification over the Ammonium Sulfate Precipitate I described in Step 1. Overall, a 60- to go-fold purification from the crude acetate extract may be obtained


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by this procedure. The results from a representative run are presented in Table I. Some of the steps in the purification procedure were adopted because of their effectiveness in removing contaminating enzymes; thus Step

TABLE I Purification of Methionine-Activating Enzyme

For the conditions of the assay, see the text.

Total units* Units per ml. Specific activityt

Acetate extract. . . . . . 1000 7.2 0.04 Ammonium Sulfate Ppt. I.. . . 800 18.2 0.23 Isoelectric Ppt.. . . 780 22.0 0.88 Ammonium Sulfate Ppt. II. 250 48 1.55 After heat treatment....................... 190 60 3.5

* 1 unit = 1 pmole of AMe formed in 30 minutes at 37”. t Units per mg. of protein.

TABLE II Enzymatic Composilion of Different Fractions of Methionine-Activating Enzyme For the conditions of the assay of MAE, see the text; for the measurement of

LPPase activity, 3 pmoles of PP were added in place of ATP; for the measurement of ATPase activity, L-methionine was omitted from the reaction mixture.

Specific act~ivi~iuenits per mg. Ratios

MAE MAEt LPPase ATPase &I (b) (cl -a

(a) w (6) (4 Cd (f) -- --

Ammonium Sulfate Ppt. I. . . . 0.24 0.26 0.84 1.18 0.3 2.2 IsoelectricPpt . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.59 0.69 0.18 0.35 3.8 1.9 Ammonium Sulfate Ppt. II. . . . 1.0 1.39 0.1 0.42 13.9 3.2 After heat treatment. . . . . . . 2.28 2.75 0.12 0.05 23.0 55.0

* MAE, 1 unit = 1 pmole of AMe formed in 30 minutes at 37”; LPPase, 1 unit = 1 rmole of pyrophosphate hydrolyzed in 30 minutes at 37”; ATPase, 1 unit = 1 Hmole of orthophosphate formed from ATP in 30 minutes at 37”.

t Measured with added YPPase (30 units with 3 y of protein).

2, isoelectric precipitation, achieves the removal of most of the LPPase (Table II, Column e), and Step 4 removes almost completely the residual ATPase (Table II, Column f).

Other Enzyme Preparations--Preparations of crystalline and highly purified pyrophosphatase were obtained initially through the generosity of Dr. M. Kunitz and Dr. Leon Heppel, respectively, and later prepared from dried bakers’, yeast by the procedure of Heppel and Hilmoe (6). Potato


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apyrase was prepared according to Krishnan (7). Myokinase was prepared according to Colowick and Kalckar (8).

Measurement of Enzyme Activity-The activity of the MAE can be determined either by measuring the amount of IP liberated from ATP in the presence and absence of methionine (1) or by determination of AMe directly (9).

MAE activity was measured in a reaction mixture of the following composition: 0.02 M ATP; 0.02 M L-methionine; 0.008 M GSH; 0.3 M MgCL; and 0.13 M Tris buffer at pH 7.6. The cold enzyme solution was added to the reaction mixture at room temperature and incubated at 37” for 30 minutes.

Under the conditions of the assay, the formation of AMe was approximately linear with time and proportional to enzyme concentration. The reaction was terminated by the addition of 6 per cent perchloric acid, and aliquots of the protein-free filtrate were used for the determination of IP (10) and AMe.

The determination of AMe is based on the fact that this compound, on account of its cationic nature, is not adsorbed on an anion exchange resin (Dowex 1 chloride) under conditions favoring the absorption of most other adenine derivatives. Adenosine is not adsorbed, but this fact does not vitiate the validity of the method, since adenosine is not formed enzymat- ically from ATP under the conditions of this study. Specifically the procedure was as follows: An aliquot of the perchloric acid filtrate containing 3 to 5 pmoles of adenine nucleotides was transferred to a graduated cylin- der containing about 15 ml. of Tris buffer (0.015 M, pH 7.4) and 1 drop of brom thymol blue (0.04 per cent). After carefully adjusting the pH to 7.4 by dropwise addition of 1.0 N NaOH, 2 ml. of a 50 per cent suspension of Dowex 1 chloride (X-10, 200 to 400 mesh) were added, and the volume was brought to 25 ml. The contents were mixed repeatedly by inversion during the next 15 minutes, and after filtration the optical density of the solution was read at 260 rnp in the Beckman DU spectrophotometer. A molar extinction coefficient of 16,000 has been used to calculate the AMe concentration. PP was determined as easily hydrolyzable phosphate after removal of adenine nucleotides with Norit A (11).

Results

Formation of Pyrophosphate-According to Equation 1, the ratio of IP to AMe should be 3. Such a ratio was invariably observed with crude preparations of the MAE. However, during the course of enzyme purification it became evident that the ratio of IP to AMe decreased progressively and in the best fractions approached a value of 1 (Table III). Further-. more, losses in total activity were encountered during the fractionation


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procedures, and such losses were particularly apparent when the activity was measured by the phosphate method. As indicated previously (4), examination of the enzymatic pattern of different fractions revealed the fact that crude MAE contained from 5 to 15 units of LPPase per unit of MAE, while in purified fractions the ratio of LPPase to MAE was as low as 0.05 (Table II).

These observations were interpreted as an indication that, in the course of the activation reaction, PP was formed, and furthermore that it accumu- lated, in the absence of sufficient LPPase, in amounts large enough to inhibit the synthesis of AMe (Table II, Columns a and a). If this interpre- tation is correct, a more precise formulation of the activation reaction may

TABLE III Effect of Enzyme Pu@cation on Formation of AMe and IP

The experimental conditions were identical with those described for assay in the text. When indicated, 3 y of YPPase (30 units) were added per ml. of solution.

AMe IP

/moles pmoles

Ammonium Sulfate Ppt. I .................. 1.50 4.39 “ ‘I “ I + YPPase ....... 1.6 4.8

Isoelectric Ppt ............................ 1.56 2.03 Ammonium Sulfate Ppt. II ................. 2.8 2.85

“ ‘I “ II + YPPase ...... 3.2 9.4

IP AMe

2.9 3.0 1.3 1.03 2.94

be represented by Equations 2 and 3, the sum of which is equal to Equa- tion 1 above.

(2) L-Methionine + ATP GSH

A AMe + PP + IP Mg++

(3) PP --=I-+ 21P WP

(2) + (3). GSH

L-Methionine + ATP A WP+

AMe + 31P

Three lines of experimental evidence were obtained in support of this formulation: (a) Addition of YPPase stimulated the formation of AMe and restored the ratio of IP to AMe to values approaching 3.0 (Table III). The effect of YPPase on the formation of AMe was reflected in a parallel increase in the disappearance of ATP, determined by the procedure of Cohn and Carter ((12) ; Table IV) ; (b) in the absence of added YPPase,


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it was found that AMe, IP, and PP were formed in approximately equivalent amounts (Table V). The deviations from the values which would be expected for Equation 2 are small and can probably be accounted for by the fact that the purified MAE is still not entirely free of contaminating

TABLE IV E$ect of Addition of PPase on AMe Formation and on ATP Disappearance

The experimental conditions were identical with those defined for MAE assay in the text, except that less ATP was used, and that methionine and 3 y of YPPase (30 units) svere included or omitted as indicated. The results are expressed in per cent of total adenine nucleotides. The actual values of the total adenine nucleotide in the four samples were 12.12, 11.95, 11.84, and 11.75 pmoles of adenine, respectively.

YPPase added No YPPase

Nucleotide M&hi&m Methionine

-t- A + - A __________~_______

AMe . . . . . . . . . . . 34.6 1.27 +33.3 17.8 1.19 +16.6 AMP . . . . . . . . . . . . . . . . . . 4.1 2.01 +2.1 3.28 2.05 +1.23 ADP . . . . . . . . . . . 10.4 11.7 -1.3 11.0 11.8 -0.8 ATP . . . . . . . . . . . . . . . . . 50.8 85.5 -34.2 67.8 85 -17.2

TABLE V Equivalence of S-Adenosylmethionine, Pyrophosphate, and Orthophosphate Formation

The experimental conditions were identical with those defined for the MAE assay in the text.

Experiment No.

la lb 2a 2b*

Incubation time

tnin.

30 60 30 30

AMe IP

(a) (b)

~m01.3 p?iWh

2.92 3.50 4.2 5.02 2.06 2.55 2.46 7.64

PP

(4

pmoles

3.1 3.9 2.25 0.06

(b) 73

(a)

1.12 1.2 1.2 3.1

(c) (a)

(4

1.06 0.93 1.09 0

* 20 units of purified YPPase with 2 y of protein were added.

LPPase; (c) PP was found to inhibit the formation of AMe. It is noteworthy that there is a marked effect of pH on this inhibition; at low con- centrations of PP the inhibition is evident only at pH levels below 7.0. This might indicate that the true inhibitor is the pyrophosphate anion H2P20,“, since this species is the predominant one at pH levels below 6.8.

Origin of Pyrophosphate-In order to gain an insight into the reaction mechanism, it was thought desirable to determine which two of the three


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phosphates of ATP2 give rise to PP. For this purpose ATP labeled with P32 was used, and its specific activity compared with that of the PP formed during the reaction.

In the first experiment (Table VI) vessels with 3 ml. of a complete reaction mixture containing ARP-P32-P32 were incubated for 20 and 40 minutes, respectively, at 37”. The reaction was terminated by the addition of 4 ml. of 6 per cent perchloric acid. Control reaction mixtures without L-methionine were also incubated; in addition, for initial values identical reaction mixtures were set up, and perchloric acid was added before the

TABLE VI Origin of Inorganic Pyrophosphate As Studied by Means of ARP-Pas-Pa2

Each vessel contained 22.5 pmoles of ATP, 15 pmoles of glutathione, 400 rmoles of MgC$, 200 pmoles of Tris-HCl buffer, pH 7.4, and 15 mg. of protein in a final volume of 3.0 ml. 30 pmoles of L-methionine were added to the complete system. Specific activities of inorganic phosphate and pyrophosphate are corrected for zero time values.

Zero time Complete

“ No methionine I‘ ‘I

Specific activity

-7 Incubation time

I I PPy IP*

min.

20

_.

-

ATP$

5360 5220 5420

5000

* Counts per minute per micromole of phosphate. t To correct for the addition of carrier PP, the values in this column were ob-

tained by multiplying the measured values by the appropriate dilution factor. $ Counts per minute per micromole of labile phosphate.

addition of the MAE. After centrifugation 2 ml. of water and 200 mg. of Norit A were added to a 5 ml. aliquot of the protein-free supernatant fluid, and the suspension was clarified by centrifugation and filtration (10) ; IP and PP were then determined on small aliquots of the filtrate. A larger aliquot of the filtrate, plus 5.3 hmoles of carrier PP, was adjusted to pH 8.2 and chromatographed through a Dowex 1 Cl column (X-10, 200 to 400 mesh). The column was first washed with 25 ml. of water and then with 100 ml. of 0.01 N HCl to elute IP. The effluent was collected in four 25

2 In the text, the phosphates of ATP will be designated as (Y-, p-, and y- as follows: ARP-P-P

1 1 1 ci BY


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ml. fractions; most of the IP appeared in the first 25 ml. fraction, while the third and fourth fractions were entirely free of radioactivity. The column was then eluted with 0.01 N HCl in 0.2 M NaCl and the effluent collected in 5 ml. fractions; PP, as measured both by radioactivity and by determinations of easily hydrolyzable phosphate on aliquots of the eluate, appeared sharply in the second, third, and fourth fractions. The specific activity of the p- and y-phosphates of the ATP was obtained as follows: The Norit residue was washed three times with 10 ml. of water and then suspended in 5 ml. of 1 N HCl, maintained for 10 minutes at 100” to hydro- lyze the labile phosphate, cooled, and centrifuged, and the supernatant fluid was decanted and the specific activity of the IP was determined on an aliquot.

The results of this experiment (Table VI) clearly show that the PP formed in the reaction catalyzed by the MAE had a specific activity almost exactly one-half that of the IP derived by acid hydrolysis from the ,B- and y-phosphates of ATP, whereas the specific activity of the IP formed in the activation reaction approached that of the r-phosphate in ATP. The values found differ markedly from those which would be expected if the PP were derived from the two terminal phosphates in ATP (theoretical: IP, 0; PP, 5420). On the other hand, the data agree closely with the predictions from a mechanism requiring that PP formed in the course of the activation reaction be derived from the o(- and ,&phosphates of ATP (theoretical: IP, 5420; PP, 2710). The deviations from the theoretical are small and con- sistent with the presence of residual LPPase in the enzyme preparation.

These results were confirmed by a second experiment, essentially identical in design to the one just described but different in that ARP32-P-P was used in the reaction and no carrier PP was added before chromatography. To determine the specific activity of the phosphates of ATP separately, first the specific activity of the labile phosphate was determined as in the preceding experiment; second, chromatographically pure ATP was obtained, and the specific activity (counts per minute per micromole of ATP) of the entire molecule, assuming a molar extinction coefficient of 16,000 at 260 rnp, was determined. The specific activity of the a-phosphate was then obtained by subtraction of the sum of the values for the p- and y- phosphates from the value for the whole molecule.

The results of this experiment, as shown in Table VII, confirm entirely the above conclusion. Again, the values differ markedly from those that would be expected if the terminal two phosphates of ATP give rise to PP (theoretical: IP, 543; PP, 21); they closely approximate ,the theoretical values (IP, 21; PP, 282) to beexpectedif the PP were derived from the a- and &phosphates of ATP. To indicate the origin of the PP molecule formed in the activation reaction, Equation 2 above can therefore be rewritten as


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GSH L-Methionine + ARP*-P*-P - Mg++

AMe + P*P* + IP

Nucleotide SpecijZty-As far as is known, only ATP will serve as a nucleotide source for the synthesis of AMe. The possible participation of ADP was investigated with particular care, since the formulation described in Equation 4 raises the question whether the .ADP moiety of ATP might function directly as an adenosine donor or might be involved as intermediate in the activation reaction.

Both possibilities appear to have been ruled out by the following experiments: ADP itself is inferior to ATP as a substrate for the formation of

TABLE VII Origin of Inorganic Pyrophosphate As Studied by Means of ARP32-P-P

The complete system contained 30 pmoles of ATP, 25 amoles of n-methionine, 450 pmoles of MgClz, 225 pmoles of.Tris-HCl buffer, pH 7.4, 7 rmoles of glutathione, and 1.1 mg. of protein (3.9 units of enzyme) in 1.5 ml. final volume. The specific activity of the a-phosphate of ATP was 543 c.p.m. per micromole; those of theg- and r-phosphates were each 21 c.p.m. per micromole of phosphate. The specific activities are corrected for zero time values.

Incubation time

min.

P* PP’

Complete . . . 15 259 “ . . . . . . . . . .._..._ 45 52 248

* Counts per minute per micromole of phosphate.

AMe. Furthermore, while AMP was not formed in appreciable amounts when ATP was used as a substrate, it was formed in nearly stoichiometric amounts with AMe when ADP was used, and this is probably due to the presence of residual myokinase in the preparation of MAE used.

Several experiments were performed with nucleotides labeled isotopically in different ways to investigate further the possible role of ADP in the formation of AMe. In the first experiment (Experiment A, Table VIII) AMe was formed eneymatically from a reaction mixture containing ATP labeled in the a-phosphate with P32 and an approximately equivalent concentration of ADP. The PP formed was isolated by chromatography3 of an aliquot of the reaction mixture (after nucleotide removal with Norit A), and its specific activity was determined on an aliquot of the eluate. The nucleotides were separated on Dowex 1 Cl (12), and their specific activities were

3 No carrier PP was added before chromatography in this experiment. PP determinations were confirmed with YPPase.


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determined on appropriate aliquots, assuming a molar extinction coefficient of 16,000 at 260 rncc. It can be seen that the specific activity of the PP compares closely with that of the ATP and not the ADP, thus indicating that ATP is the substrate for the MAE. The small increase in the labeling of ADP is probably due to residual ATPase.

TABLE VIII Non-Participation of ADP in Activation Reaction

In Experiment A, the reaction mixture contained 40 pmoles of ATP labeled with Pa2 specifically in the a-phosphate, 40 pmoles of ADP, 0.7 mmole of MgClt, 0.5 mmole of Tris-HCl buffer, pH 7.4,50pmoles of GSH, 60 pmoles of L-methionine, and 6.7 mg. of protein in a final volume of 6.2 ml. In Experiment B, the reaction mixture contained 40 pmoles of ADP labeled with Cr4, 40 amoles of ATP, 0.7 mmole of MgC&, 0.35 mmole of Tris-HCl buffer, pH 7.4,50 pmoles of GSH, 60 pmolee of n-methionine, and 5.6 mg. of protein in a final volume of 6.0 ml.

Experiment A

Time of lncubatlon Specific activities, c.p.m. per lunole

PP I ADP I ATP

Zero time.. . . . 30min.. . . . . 60 Ic .

740 780

Experiment B

25 790 50 810 75 780

Condition Specilic activities, c.p.m. per runok

ADP ATP AMe

Zero time.. . . . . . . . . . . . . . . 2575 330 0 No methionine.. . . . . . . . 1650 940 0 Complete. . . . . . . . . . . . . . . 2050 1350 875

In another similarly designed experiment (Experiment B, Table VIII), C14-labeled ADP and cold ATP were used; after the nucleotides were separated, the specific activity of the AMe formed was found to be considerably lower than that of the ADP and approximately equal to the estimated mean specific activity of the ATP during the incubation period. This Ford- ing, together with the results of the previous experiment, indicates that both the PP formed by the action of the MAE and the adenosine moiety of AMe are truly derived from ATP and not from ADP. Conversely, ADP cannot be a free intermediate. This has been corroborated in an experiment with ATP labeled in the a-phosphate with Pa2 in the presence of a


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pool of cold ADP to trap any ADP formed as a free intermediate; the formation of AMe did not result in labeling of the ADP pool.

ITP and UTP did not function as substrates for the MAE. HzO18 Experiments--The experiments described above suggested that a

compound consisting of an ADP moiety linked to methionine, specifically a phosphosulfonium, might be an intermediate in the formation of AMe. TO subject this hypothesis to a rigorous experimental test, the reaction was performed in Hz018 to investigate the points of cleavage of the poly- phosphate chain of ATP. If such an intermediate were indeed formed, it would be expected that one-seventh of the oxygen atoms in PP would be derived from the 0’“containing water of the medium, whereas IP would

TABLE IX

0’8 Distribution in IP and PP Isolated from Activation Reaction Containing HzO’s

In Experiment, 1, the reaction mixture contained 375 pmoles of L-methionine, 375 pmoles of ATP, 3.6 mmoles of Tris-HCl buffer, pH 7.4,l.g mmoles of MgC12,80 pmoles of GSH, He018, and 12 mg. of protein in a final volume of 15 ml. In Experiment, 2, the conditions were identical, except for the protein content, which was 19.6 mg.

Experiment No. Observed atom per cent excess 0’8 per atom of oxygen

&Of I

IP’ I

PP’

1 1.40 0.50 0.0 2 1.45 0.32 0.0

* Analyzed as CO.

retain its 4 oxygen atoms. Experimentally just the opposite result was observed; namely, it was found that the PP moiety retained all of its original 7 oxygen atoms, whereas 01* was incorporated into the orthophosphate (Table IX). Clearly then the terminal phosphate separates from the poly- phosphate chain as the POP= moiety, and a phosphosulfonium derivative of ADP and methionine is not possible; indeed, this experiment also rules out the possible formation of other compounds of ADP and methionine such as the acylphosphate or the phosphoamide.

Reversibility Experiments--The possibility that either the over-all reaction or one of the partial reactions might be reversible was investigated isotopically under a variety of conditions by using in separate experiments P32, P32-labeled PP, CY4-adenosine, and S35-AMe, and measuring ,the incor- poration of the isotopes into ATP and methionine, respectively. All such experiments failed to demonstrate any exchange reactions attributable to MAE.


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DISCUSSION

The results presented above are of interest from two points of view. In the first place, they reveal an entirely new mechanism for the utilization of ATP in biological systems. Heretofore ATP has been known to act (a) as a phosphate donor with the concomitant formation of ADP, (b) as a pyrophosphate donor with the concomitant formation of AMP, and (c) as an adenylate donor with the resultant mineralization of its terminal pyrophosphate moiety. While the intimate mechanism of the reaction studied here is not yet fully understood, it is clear that this reaction cannot be grouped in any of the three categories above, and furthermore it is the first demon- stration of the fact that ATP functions as an adenosine donor. The finding that PP can be formed from the CY- and &phosphates of ATP is novel and indicates that the ribose phosphate bond in ATP is not as inert as had been thought until now.

Because of the complexity of the reaction described in Equation 1, it had been expected that it might be possible to gain an insight into it by attempts at resolving the crude MAE into two or more fractions. These expectations were borne out only in part. The MAE has been purified between 60- and go-fold, and the participation of LPPase has been clearly demonstrated. However, attempts at further resolution of the MAE proper have been negative. Moreover, attempts to find an intermediate by chromatographing reaction mixtures prepared from radioactive substrates have failed.

Recently Hoagland et al. (13) and Berg (14) have discovered in rat liver and in yeast the existence of an amino acid-dependent PP-ATP exchange reaction. The general characteristics of this system and in particular the accumulation of aminoacylhydroxamic acids and PP in the presence of hydroxylamine indicate that probably carboxyl activation of the amino acid is involved, with the formation of an acyl-AMP intermediate. The activation system from rat liver has been separated into a number of different protein fractions differing only by the amino acid specificity, and it has been reported that one fraction is specific for L-methionine (13).

It is noteworthy that the purified MAE did not catalyze a methionine- activated PP-ATP exchange reaction. Moreover, a liver fraction containing the amino acid-activating system was inactive in the synthesis of AMe. Finally, hydroxylamine appears to be inert in the MAE system, since the addition of the salt-free base to the reaction mixture was without effect on the formation of AMe and did not result in the accumulation of “hydroxamic acids.” It is clear therefore that there are two enzymatic reactions involving methionine and ATP. Although the two methionine-

4 Kindly supplied by Dr. M. B. Hoagland.


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activating systems exhibit some common features, namely the requirement for ATP and the formation of PP in the course of the activation reaction, they differ in every other respect; namely, in the apparent biological purpose of the activation (transmethylation versus protein synthesis) and in some of the products (AMe versus AMP-methionine), in the origin of the PP formed (a- and @-phosphates versus ,& and y-phosphates of ATP) and presumably also in the molecular mechanism of the activation.

In order to arrive at a satisfactory formulation of the mechanism of the reaction catalyzed by MAE, it is necessary to consider the following observations: (a) the results of experiments on the origin of the PP and IP formed in the reaction, (b) the results of the Or* experiments, (c) the results of experiments indicating the lack of participation of ADP, (d) the lack of reversibility as measured by various exchanges, and finally (e) the finding that hydroxylamine is without effect in this system.

As must be apparent, the only mechanisms that can be proposed at the present time are hypothetical ones, since attempts to define a reaction mechanism that could be substantiated experimentally have not yielded positive results.

One possible formulation would require an intramolecular migration of the terminal phosphate of ATP to form a modified adenosine triphosphate. Positions 2 and 3 in the ribose or the amino group of the adenosine moiety come to mind as possible sites for this migration. It is easy to visualize how this modified ATP could then react with methionine to form AMe, PP, and IP, and to satisfy the requirements listed above. Alternatively, the intermediate formation of an adenine “cyclonucleoside” (15), followed by nucleophilic attack by methionine to yield AMe, may be entertained. It should be pointed out, however, that both of these formulations are hypothetical, and only future work will reveal whether either one of these speculations has any factual validity.

Other Preparations

Labeled PP was prepared by heating Ps2-labeled NaH2P04 to 210’ for 12 hours.

Labeled ATP was prepared by incubating aerobically 10 pmoles of AMP (or AMP32) and 25 pmoles of P32-labeled IP (or IP) with rat liver mitochon- dria in the presence of Tris buffer, Mg++, catalytic amounts of ATP and cytochrome c, and substrate amounts of cr-ketoglutarate. After deprotein- ization, AMP, ADP, and ATP were separated chromatographically on a Dowex 1 Cl column (12) and precipitated at pH 8.0 as the barium salts.

For the preparation of AMP32, ATP labeled with Ps2 in all three phos- phates5 was incubated with highly purified potato apyrase in the presence

6 Obtained from the Schwarr Laboratories, Inc.


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of magnesium. At the end of the incubation, the IP formed was removed by addition of magnesia mixture (16), and the AMP precipitated almost, quantitatively at pH 5.5 by addition of ZnClz (17). The precipitate was dissolved in 0.5 M NH40H and reprecipitated as the barium salt after chromatography through a Dowex 1 Cl column.

ADP labeled with Cl4 in the adenosine moiety was prepared from C14- AMP and ATP by incubation with myokinase, followed by chromatography on a Dowex 1 Cl column and precipitation as the barium salt,.

Prior to use, all barium salts were decomposed in the usual way with K#Oh.

SUMMARY

1. The methionine-activating enzyme has been partially purified from rabbit liver and the mechanism of the activation reaction studied.

2. It has been found that, in addition to S-adenosyhnethionine, pyrophosphate and orthophosphate are formed in stoichiometric amounts.

3. The origin of the pyrophosphate and the site of cleavage of the poly- phosphate chain of ATP have been studied by means of the isotopic tracer technique. It has been established that (a) the pyrophosphate is derived from the OL- and /%phosphates’of ATP and retains all of its original 7 oxygen atoms; (b) the orthophosphate is derived from the terminal phosphate of ATP and acquires 1 oxygen atom from the medium.

4. The significance of these findings to the mechanism of the activation reaction has been discussed.

The authors are greatly indebted to Dr. George Drysdale of Washington University, St. Louis, Missouri, for his very generous cooperation in per- forming the analyses of the 018 experiments. They also want, to express their thanks to Dr. Michael Yarmolinsky for his help in some of the experiments.

BIBLIOGRAPHY

1. Cantoni, G. L., J. Bid. Chem., 189,745 (1951). 2. Cantoni, G. L., J. Biol. Chem., 204,403 (1953). 3. Cantoni, G. L., in McElroy, W. D., and Glass, B., Phosphorus metabolism,

Baltimore, 2, 129 (1952). 4. Cantoni, G. L., Abstracts, 3rd International Congress of Biochemistry, Brussels,

233 (1955). 5. Warburg, O., and Christian, W., Biochem. Z., 310,384 (194142). 6. Heppel, L. A., and Hilmoe, R. J., J. Biol. Chem., 192, 87 (1951). 7. Krishnan, P. S., Arch. Biochem., 20, 261 (1949). 8. Colowick, S. P., and Kalckar, H. M., J. Biol. Chem., 148, 117 (1943). 9. Cantoni, G. L., in Colowick, S. P., and Kaplan, N. O., Methods in enzymology,

New York, 3, 600 (1957).


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10. Lohmann, K., and Jendrassik, L., Biochem. Z., 178, 419 (1926). 11. Crane, R. K., and Lipmann, F., J. Biol. Chem., 201, 235 (1953). 12. Cohn, W. E., and Carter, C. E., J. Am. Chem. Sot., 72,4273 (1950). 13. Hoagland, M. B., Keller, E. B., and Zamecnik, P. C., J. Biol. Chem., 218, 345

(1956). 14. Berg, P., J. Am. Chem. Sot., 77,3163 (1955). 15. Clark, V. M., Todd, A. R., and Zussman, J., .I. Chem. Sot., 2952 (1951). 16. Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometrie techniques and

related methods for the study of tissue metabolism, Minneapolis, 184 (1946). 17. Owens, R. G., Science, 122, 415 (1955).


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G. L. Cantoni and J. DurellTHE REACTION

STUDIES ON THE MECHANISM OF METHIONINE-ACTIVATING ENZYME:

TRANSMETHYLATION: II. THE ACTIVATION OF METHIONINE FOR

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