ACYL ADENYLATES: THE INTERACTION OF ADENOSINE TRIPHOSPHATE AND 1L-METHIONINE* By PAUL BERG{ Wits THE TECHNICAL ASSISTANCE OF GEORGIA NEWTON (From the Department of Micrebiology, Washington University School of Medicine, St. Louis, Missouri) (Received for publication, March 9, 1956) Several reactions are now known which can account for the exchange of PP with the terminal pyrophosphate group of ATP (1-6). Recently it was demonstrated (3, 4) that the reaction of ATP and acetate with the acetate-activating enzyme (aceto-CoA-kinase) resulted in such an exchange. Because of this and other observations (4), it was proposed that the acetate- dependent exchange occurred via the intermediate formation of adenyl acetate (Reaction 1). (1) ATP + acetate = adeny) acetate + PP Further investigation of PP-ATP exchange reactions revealed that yeast extract catalyzed such an exchange, which was dependent on the presence of t-methionine (3). The possibility that this represented an activation of methionine by ATP, analogous to that shown in Reaction 1, stimulated further study of its mech- anism. The present report is concerned with the partial purification of an enzyme from yeast which carries out the L-methionine-dependent exchange of P?P# and ATP, and a description of some of the properties of the reac- tion. In the presence of ATP, t-methionine, and hydroxylamine there is a net formation of A5P, PP, and methionine hydroxamic acid. These find- ings, together with the failure to observe any exchange of A5P-C™ with ATP in the presence of methionine, are consistent with the formation of an adenyl methionine derivative from ATP and methionine (Reaction 2) and its subsequent cleavage in the presence of hydroxylamine (Reaction 3). (2) ATP + methionine = adenyl methionine + PP (3) Adenyl methionine + hydroxylamine — methionine hydroxamate + A5P + PP * This work was supported by granta from the United States Public Health Serv- ice and the National Science Foundation. { Scholar in Cancer Research of the American Cancer Society. 1 The following abbreviations have been used. PP, inorganic pyrophosphate; ATP or ARPPP, adenosine triphosphate; A5P, adenosine-5’-phosphate; TCA, tri- chloroacetic acid; Tris, tris(hydroxymethyl)aminomethane; CoA, coenzyme A. 1025 1026 ATP-METHIONINE REACTIONS Hoagland (5) has reported the existence of amino acid-requiring PP-ATP exchange reactions in liver extracts and demonstrated the formation of amino acid hydroxamates from ATP, amino acids, hydroxylamine, and a soluble protein fraction from liver. DeMoss and Novelli (6) have also demonstrated the existence of a number of amino acid-dependent PP-ATP exchange reactions in a variety of bacterial extracts. More recently Hoag- land et al. (7) reported the separation of the enzymatic activity responsible for the methionine-activated PP-ATP exchange reaction from other amino acid-requiring PP-ATP exchange systems and have suggested that each of the exchange reactions is catalyzed by a separate enzyme. Materials and Methods PP was prepared by heating Na,HP#O, at 225° for 18 hours (8), or at 400° for 1 hour (9), and purified by anion exchange chromatography (8). ATP labeled with P** in the terminal pyrophosphate group was prepared by exchange of P#P® with ATP by using purified aceto-CoA-kinase (4). A5P-C™ was made as previously described (4). A5P deaminase was ob- tained from rabbit muscle by the method of Kalckar (10), and crystalline ribonuclease was obtained from the Worthington Biochemical Corporation. Hydroxylamine was prepared from hydroxylamine sulfate and barium hy- droxide (4), and pt-methionine hydroxamate? was made by treatment of pL-methionine hydrochloride ethyl ester (11) with methanolic hydroxyl- amine in sodium methoxide (12) and recrystallized three times from meth- anol-water mixtures at about pH 8. Methionine hydroxamic acid was determined colorimetrically as the ferric complex (13). To the sample in a volume of 1 ml. was added 0.5 ml. of a 10 per cent solution of ferric chloride containing 0.2 m TCA and 0.66 m HCl. After 5 minutes the optical density at 540 my was determined in a cuvette with a 1 cm. light path. 1 umole of methionine hydroxamic acid gave, under these conditions, an optical density of 0.437. In the experiments in which the enzymatic formation of methi- onine hydroxamate was measured, synthetic methionine hydroxamate was added to a control tube (minus ATP and methionine) as an internal stand- ard. A5P was determined either by anion exchange chromatography (14) or by A5P deaminase (15), and PP was measured by phosphate liberation (16) after treatment with inorganic pyrophosphatase® (17). Protein was determined as described by Lowry et al. (18). ?I am deeply indebted to Dr. Peter H. Lowy, to Dr. E. B. Keller, and Dr. M. B. Hoagland for their generous gifts of pi-methionine hydroxamate which were used for comparison with the preparation described above. 3 The crystalline inorganic pyrophosphatase, prepared from yeast, was very kindly supplied by Dr. G. Perlmann and Dr. M. Kunitz. P, BERG 1027 Assay Procedure—The methionine-dependent P*P*”-ATP exchange reac- tion was measured in the following way. The reaction mixture contained, in 1.0 ml., 0.1 M Tris buffer, pH 8.0, 0.002 m ATP, 0.002 m P?P® containing between 10‘ and 10° c.p.m. per umole, 0.003 M L-methionine, 0.005 M MgCl, and the enzyme. After 15 minutes at 37°, perchloric acid was added and the P®* incorporated into ATP was measured by adsorption and elution of the ATP from Norit (4). 1 unit of activity was defined as that amount of enzyme which catalyzed the incorporation of 1 umole of P?P® into ATP in 15 minutes. With 0.3 unit of enzyme or less, the rate of exchange was constant for at least 30 minutes. By the standard assay, the amount of exchange was proportional to enzyme concentration. Thus, with 1.5, 3.0, 5.0, 12.5, and 25 y of enzyme protein (Fraction AS-1) the number of units of activity per mg. of protein were 6.7, 6.7, 6.0, 5.6, and 6.4. Results Dried brewers’ yeast‘ (25 gm.) was mixed with 75 ml. of 0.1 Mm potassium bicarbonate and incubated at 37° for 4 hours. The autolyzed mixture was centrifuged at 10,000 * g for 10 minutes and the residue discarded. The supernatant fluid (yeast extract (Fraction YE) 40 ml.) was diluted to 120 ml. with cold water, and 76 ml. of ethanol at 4° were added at a rate ad- justed to maintain the temperature between 7-10°. The solution was cen- trifuged at 4° for 5 minutes at 10,000 < g and the supernatant fluid dis- carded. The precipitate was extracted with 54 ml. of 0.05 m Tris buffer, pH 8.0, and the insoluble material obtained by centrifugation was dis- carded. To the supernatant fluid were added 27 ml. of 0.5 m potassium succinate buffer, pH 6.0, the solution was cooled to 0°, and 23.5 ml. of eth- anol at —15° were added while the temperature was kept between 0-1°. The precipitate was removed by centrifugation for 5 minutes at 10,000 X g and then dissolved in 40 ml. of 0.05 m Tris buffer, pH 8.0 (Alcohol Frac- tion 2). To this solution were added 13.4 gm. of ammonium sulfate and, after 5 minutes, the mixture was centrifuged as mentioned above and the precipi- tate discarded. ‘To the supernatant fluid were added 5.0 gm. of ammonium sulfate, and, after 5 minutes, the solution was centrifuged. The precipitate was dissolved in 10 ml. of Tris buffer, pH 8.0 (Ammonium sulfate, Fraction 1 (AS-1)). This solution was then diluted with the Tris buffer to a protein concentra- tion of 2.0 to 2.5 mg. per ml. In the experiment in Table I, the volume was adjusted to 17.5 ml. and, after being warmed to 20°, 1.5 mg. of crystal- line ribonuclease in 0.3 ml. of water were added. After 5 minutes the solu- ‘ Dried brewers’ yeast, strain BSC, was kindly furnished by Anheuser-Busch, Inc., St. Louis. 1028 ATP-METHIONINE REACTIONS tion was cooled to 0° and 31 ml. of cold saturated ammonium sulfate were added. After another 5 minutes the precipitate was removed by centrifu- gation for 10 minutes at 10,000 x g. To the supernatant fluid were added 20 ml. of saturated ammonium sulfate, and after 5 minutes the precipitate was centrifuged, as described above, and dissolved in 6 ml. of 0.05 m Tris buffer, pH 8.0 (Ammonium sulfate, Fraction 2 (AS-2)). Tass I Purification of Enzyme Concentra- Concentra- n : e Specific Fraction oon of Total units tion of activity units per ml. mg. per ml. i. be Yeast extract (YE)..................., 16.9 676 50.7 0.33 Aleohol fraction 2 (A-2)................ 15.2 608 5.0 3.0 Ammonium sulfate Fraction 1 (AS-1) ..| 34.0 340 4.1 8.3 “ “e “ 2 (AS-2) .. 28.4 170 1.9 15.0 TaBLeE II Requirements for Exchange of P#*P** with ATP Components Pi1p3t incorporated into ATP umole Complete... ........0.. 00.00 cee eee 0.38 No ATP....... 000. eee 0.01 “ methionine..............0..... 0020005 0.03 MagCle........0..0.0 0002 eee 0.01 © emzyme.............0 0000... ee ee eee 0.00 The conditions were the same as those described for the assay of the enzyme. 50 y of enzyme Fraction AS-1, specific activity 7.5, were used. Fraction AS-2 lost about 30 per cent of its initial activity in 1 week when stored at —15°. In all of the experiments reported here, Fraction AS-1, which was more stable, was used. Fraction AS-1 still contained some ATP-splitting activity but no detectable inorganic pyrophosphatase (17) or adenylic kinase (19). Requirements for PP-ATP Exchange-—In the crude yeast extract there was little or no increase in the rate of the exchange reaction upon the ad- dition of methionine. With the purified fractions (Ammonium sulfate, Fractions 1 and 2) little or no exchange of PP and ATP occurred unless L-methionine and Mg++ were added (Table II). Most preparations of the enzyme still contained some activity in the absence of added methionine, P. BERG 1029 but this rarely exceeded 10 per cent of the rate obtained in the presence of optimal amounts of methionine. The amount of methionine necessary to promote the maximal rate of exchange was 1 X 10-4 M, and for half maximal rate it was 1 K 107-5 m (Table III). It might be pointed out that this procedure offers a relatively simple and rapid method for detecting and determining small amounts of L-methionine. Specificity of u-Methtonine Reguirement—Of the naturally occurring amino acids, only methionine catalyzed a significant amount of exchange of P#P” and ATP with Fraction AS-1 (Table IV). Other amino acids alone or in various combinations gave values no higher than the control Tas.x III Effect of u-Methionine Concentration on Rate of PP-ATP Exchange Reaction Concentration of t-methionine Pp? incorporated into ATP x 104 a umole 0.04 0.11 0.17 0.19 0.30 0.39 0.39 0.39 0.41 Fo" Ss588 AN wm SOS -_ The conditions were the same as those described for the assay of the enzyme. 50 y of enzyme Fraction AS-1, specific activity 7.5, were used. with nothing added. Furthermore, other amino acids, alone or in combi- nation, did not inhibit the methionine-activated exchange. The require- ment for methionine was found to be specific for the Lform, p-Methionine was inactive, and did not inhibit the effect of L-methionine when both were present at equal concentrations (1 X 10-?M). Methionine sulfoxide, me- thionine sulfone, and homocystine were also inactive. The only other amino acid which has been found to promote the exchange reaction was pDL-ethionine, but, because extremely small amounts of methionine are ac- tive, the exchange found with ethionine may be due to contamination with methionine. Formation of Methionine Hydroxamic Acid—Because the methionine-ac- tivated PP-ATP exchange reaction appeared to be somewhat analogous to the acetate-dependent exchange reaction by aceto-CoA-kinase (3, 4), ex- periments were carried out to detect the enzymatic formation of methionine 1030 ATP-METHIONINE REACTIONS hydroxamate in the presence of hydroxylamine. Incubation of ATP, me- thionine, Mgt*, hydroxylamine, and the enzyme resulted in the formation of equivalent amounts of methionine hydroxamic acid, A5P, and PP when the values in the absence of methionine were subtracted (Table V). Under these conditions, there was an almost linear rate of methionine TABLE IV Effect of Various Amino Acids on PP-ATP Exchange Amino acid Pps? incorporated into ATP umole None..............--....05: 0.01 t-Methionine............... 1 x 10%M 0.13 L-Tryptophan. ............. 1 xX 10°33 “ 0.01 pL-Alanine...............-. 1 & 10-3 0.01 L-Histidine................. 1 xX 10% * 0.02 L-Glutamic acid............ 1 xX107* 0.01 L-Isoleucine................ 1 x 10°33 “ 0.01 DL-Serine................... 1 x<107%* 0.00 u-Phenylalanine............ 1 «10° * 0.01 u-Valing................000. 1 x 1073 * 0,02 L-Threonine................ I & 107 « 0.02 L-Leucine.................. 1 xX 10-3 * 0.01 L-Proline...............0005 1 xX 10°“ 0.01 pL-Homocysteine........... 1 x 103“ 0.01 Glycine...........00.0...0., 1 ™& 1073 0.01 L-Tyrosine.................. 1 X* 10% * 0.02 None... 0.0... ees 0.04 u-Methionine............... 1.5 xX 107° m 0.41 p-Methionine............... 1 «10°73 * 0,05 (6 been e tee eeees 3 X10°3 ‘ 0.04 pu-Ethionine............... 3 xX 1073 0.12 Both experiments were carried out as described for the usual assay procedure. In the first experiment, 12 y of enzyme Fraction AS-1, specific activity 10, were used, and in the second experiment 50 y of Fraction AS-1, specific activity 7.5. hydroxamic acid and PP formation. However, this rate decreased rapidly after 60 minutes and was not restored by the addition of more ATP and methionine. The reason for this is not clear. When the enzyme, ATP, Mgt+, or methionine was omitted, there was no significant formation of methionine hydroxamic acid above that ob- served in the absence of methionine. The absorption spectrum of the enzymatically produced methionine hydroxamate in the presence of ferric chloride at acid pH was characteristic of acyl hydroxamic acids. This spectrum exhibited a broad maximum between 495 and 510 mu. P. BERG 1031 Nature of Reaction of ATP and Methtonine—Two possible interpretations of the mechanism of the reaction were considered. In the first, ATP and methionine react to form a methionine pyrophosphate compound which can subsequently exchange the bound pyrophosphate group for free P?P* (Reactions 4 and 5). In the second, the products formed are adenyl me- TABLE V Enzymatic Formation of Methionine Hydrozamate, A&P, and PP from ATP, Methionine, and Hydrorylamine The values are given in micromoles per ml. Experiment! Time Additions ASP a’ PP ae nae hy a . droxamate min. 1 30 Complete 1.47 | +1.383 | 1.24 | +1.19 60 “ 2.17 | +1.90 | 2.00 | +1.93 30 No methionine 0.14 0.05 60 “ 7 0.27 0.07 2 30 Complete 1.79 | +1.63 | 1.81 | +1.72 | 0.97 +0.88 30 No methionine | 0.95 | +0.79 | 0.99 | +0.90]} 0.09 0.00 30 ‘ATP 0.16 0.09 0.09 *The difference was calculated by subtracting the values obtained in the ab- sence of either methione or ATP. Experiment 1. The reaction mixture (1.0 ml.) contained 0.10 m Tris buffer, pH 8.0, 0.005 m MgClo, 0.011 m ATP, 0.01 mw u-methionine, 2.5 m hydroxylamine, and 1.2 mg. of Fraction AS-1, specific activity 12.1. After incubation at 37°, aliquots were removed for measurement of methionine hydroxamic acid formation as previously described, and PP was determined after conversion to inorganic phosphate with in- organic pyrophosphatase. Experiment 2. The reaction mixture (1.15 ml.) con- tained 0.09 m Tris buffer, pH 8.0, 0.004 m MgCl:, 0.011 m ATP* containing 5400 c.p.m. per ymole, 0.01 mM L-methionine, 2.2 m hydroxylamine, and 1.2 mg. of Fraction AS-1, specific activity 11.4. After incubation at 37°, aliquots were measured for methio- nine hydroxamate as described previously. One series of aliquots was acidified and treated with Norit (4), and the PP was calculated from the P*? in the nucleo- tide-free supernatant fluid. Separate aliquots were chromatographed on Dowex 1 to separate the ASP, and the total amount of A5P was determined from the op- tical density at 260 my, thionine and free PP (Reaction 6), which by reversal of the reaction con- verts PP? to ATP®. (4) ATP + t-methionine = L-methionine-PP + A5P (5) t-Methionine-PP + P??P!* = t-methionine-P#P2? + PP (6) ATP + t-methionine = adeny! L-methionine + PP To distinguish between these two possibilities, the exchange of A5P-C'* 1032 ATP-METHIONINE REACTIONS and ATP was studied. By mechanism (1), A5P-C"™ should exchange with ATP at least as rapidly as does P*P*, and this should require L-methionine. By mechanism (2), there should be no exchange. It was found (Table VJ) TaBLe VI Exchange of A&P-C' and ATP | Specific activity of compound isolated Components | ASP* | ATP* | c.p.m. per umole —— cpm. per mole I i Complete.................. 15, 000 <100 No methionine............., 15,500 : <100 * enzyme................ 15,000 : <100 * No correction for the self-absorption due to the salt of the eluate was made. The reaction mixture contained in 1 ml. of 0.14 m Tris buffer, pH 8.0, 0.005 x MgCl, 0.001 m ATP, 0.001 m A5P-C™ containing 2.25 X 10¢ c.p.m. per umole, and 17 y of Fraction AS-1, specific activity 6.7, Time 30 minutes; temperature 37°. At the end of the incubation, ASP and ATP were separated by anion exchange chroma- tography (14) and the radioactivity of an aliquot of the eluate from the peak tube was determined. TaBLeE VII Formation of PP from ATP P22 incorporated into PP (pa: incorporated into ATP| Labeled substrate | Time a oo S Plus Minus Plus. Minus methionine | methionine methionine | methionine ' ~~ min. umole umole umole pmole | ARPP?3P32 15 0.099 0.028 | +0.071 380 0.191 0.060 | +0.131 pszpaz 15 0.089 0.016 | +0.073 30 0.157 | 0.020 | +0.137 The reaction mixture contained, in 1 ml.,0.16 m Tris buffer, pH 8.0; 0.005 1 MgCi.; 0.001 m ATP or ATP? (ARPP#P*) containing 4.35 X 10‘ c.p.m. per umole; 0.005 n L- methionine; 0.001 m PP or P#P32 containing 3.28 X 104c.p.m, per wmole; 15 y of Frac- tion AS-1, specific activity 4.8. Temperature 37°. P®? incorporated into ATP was determined as in the usual assay procedure and P®#? into PP was measured in the su- pernatant fluid after adsorption of the ATP* with Norit. that there was little or no incorporation of A5P-C™ into ATP either in the presence or absence of t-methionine. In a comparable experiment in which 1 umole of P??P3? replaced the A5P-C"™, there was an incorporation of 0.22 umole of PP into ATP, or about 44 per cent of the maximal exchange pos- sible. This evidence would then appear to exclude the first hypothesis shown in Reactions 4 and 5. Pr, BERG 1033 Several attempts have been made to demonstrate a net formation of PP from ATP in the presence of t-methionine and in the absence of hy- droxylamine, but these have been unsuccessful to date. ATP labeled with P® in the pyrophosphate group was incubated with the enzyme, Mgt", and inorganic pyrophosphatase in the presence and absence of t-methi- onine and P® inorganic phosphate was measured in the Norit-non-adsorb- able fraction. ‘There was no significant difference in the amount of P™ liberated in the presence or absence of L-methionine. However, if in place of the inorganic pyrophosphatase a pool of unlabeled PP was added, con- siderable radioactivity from the ATP was trapped in the PP and could be determined in the Norit-non-adsorbable fraction (Table VII). The pyro- phosphate liberated was equivalent to the amount of P®#P* incorporated into ATP in a parallel experiment with PP and unlabeled ATP. Thus it is clear that, while the enzyme catalyzes the formation of free PP, the reaction does not appear to proceed very far in this direction. Whether this is due to the formation of an enzyme-bound, adenyl methi- onine compound, as had been previously suggested (5, 7), remains to be established. DISCUSSION The results obtained in the present work with L-methionine show certain similarities to those obtained with the acetate-activating system (aceto- CoA-kinase) from yeast. In both there is a reaction of ATP with a com- pound containing an acyl group, which results in the exchange of the pyro- phosphoryl group of ATP with free PP. The A5P moiety of the ATP remains in a “bound” form; that is, not exchangeable with free A5P. Furthermore, in both systems the formation of any product of the reaction, except by exchange reactions or by the use of high concentrations of hy- droxylamine, has not been demonstrated. The formation of methionine hydroxamate suggests that the methionine is linked through the carboxyl group, presumably to the phosphate group of A5P. One notable difference between the acetate-activating system and the present reaction with methionine is the subsequent transfer of the acetyl group to an acceptor (CoA). This poses the question of what is the nat- ural acceptor of the methionine moiety. Several attempts were made to detect an acceptor in crude yeast extracts by measuring PP liberation from ATP in the presence of the enzyme, methionine, and crude yeast fractions. There was, however, no formation of PP which could not be accounted for by the activity in the crude extracts of ATPase. Hoagland et al. (7) have suggested the possibility that the ‘“‘protein-forming site” functions as the acceptor and that the amino acids activated in this way are linked together to form the polypeptide structure. Maas (20), in his studies of pantothenic acid synthesis, has proposed that an adeny] pantoate derivative is formed 1034 ATP-METHIONINE REACTIONS from ATP and pantoic acid and that the pantoyl group is transferred to the acceptor amine f-alanine. The question of whether adenyl amino acids are enzymatically utilized for protein formation in the absence of an energy source requires further work, however. SUMMARY An enzyme has been isolated from yeast which catalyzes an L-methi- onine-dependent exchange of P#P® with ATP. p-Methionine and other amino acids alone or in combination do not replace the function of L-me- thionine. In the presence of ATP, t-methionine hydroxylamine, and the enzyme, there is a net formation of methionine hydroxamic acid, A5P, and PP. This, plus the fact that A5P-C™ does not exchange with ATP, suggests that the PP-ATP exchange can be accounted for by the following reaction: ATP + .L-methionine = adenyl L-methionine + PP BIBLIOGRAPHY . Kornberg, A., J. Biol. 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