ACYL ADENYLATES: THE INTERACTION OF ADENOSINE TRIPHOSPHATE AND L-METHIONINE* BY PAUL BERGf WITH THE TECHNICAL ASSISTANCE OF GEORQIA NEWTON (From the Department of Microbiology, Washington University SchooE of Medicine, St. Loui~, Missouri) (Received for publication, March 9, 1956) Several reactions are now known which can account for the exchange of PI" 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 1 ATP + ucetatc G adenyl ncctutc -I- PP Further investigation of PP-ATP exchange reactions revealed that yeast extract catalyzed such an exchange, which was dependent on the presence of L-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 P32P*2 and ATP, and a description of some of the properties of the reac- tion. In the presence of ATP, L-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-C14 with ATP in the presence of methionine, are consistent wit,h 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) (3) Adenyl methionine + hydroxylamine -+ ATP + methionine S adenyl methionine + PI' methionine hydroxamute + A5P + PP * This work was supported by grants from the United Statee Public Health Serv- 7 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 (hydroxymethy1)aminomethane; CoA, coenzyme A. 1025 ice and the National Science Foundation. 1026 ATP-METHIOXINE REACTIOSR 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 Sovelli (6) have also demonstrated the existence of a number of amino acid-dependcnt 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 Papa2 wy&5 prepared by heating Na2HPa20r at 225" for 18 hours (8), or at 400" for 1 hour (O), and purified by anion exchange chromatography (8). ATP labeled with Pa* in the terminal pyrophosphate group was prepared by exchange of PazP3* with ATP by using purified aceto-CoA-kinase (4). A5P-C" was made as previously described (4). A5P deaminase wm ob- tained from rabbit muscle by the method of Kalckar (lo), and crystalline ribonuclease was obtained from the Worthington Biochemical Corporation. Hydroxylamine waa prepared from hydroxylamine sulfate and barium hy- droxide (4), and DL-methionine hydroxamate? was made by treatment of DL-methionine hydrochloride ethyl ester (1 1) 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 HC1. After 5 minutes the optical density at 540 mp was determined in a cuvette with a 1 cm. light path. 1 pmole 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 tvns measured by phosphate liberation (16) after treatment with inorganic pyrophosphatase3 (17). Protein was determined as described by Lowry ct al. (18). * I nm deeply indebted to Dr. Peter H. Lowy, to Dr. 14. B. Keller, nnd Dr. M. 13. Hoaglnnd for their gcncrous gift8 of DI,-mcthionine hydroxarnnte which were used for comparison with the prepar:ttion dcvcribed shove. .I The crystallirie inorganic pyrophosphatase, prepared from yeast, was very kindly supplied by Dr. G. Perlmann and Dr. hl. Kunitz. P. BERG 1027 Assay Procedure-The methionine-dependent P32P3z-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 P32P32 containing between lo4 and 106 c.p.m. per pmole, 0.003 M L-methionine, 0.005 M MgC12, and the enzyme. After 15 minutes at 37", perchloric acid was added and the P32 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 pmole of P32P3e 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) thenumber 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 M potassium bicarbonate and incubated at 37" for 4 hours. The autolyzed mixture was centrifuged at 10,OOO x g for 10 minutes and the residuediscarded. 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 wm cen- trifuged at 4' for 5 minutes at 10,OOO X 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 O", and 23.5 ml. of eth- anol at -15" were added while the temperature was kept between 0-1". The precipitate wa~ removed by centrifugation for 5 minutes at 10,OOO X g and then dissolved in 40 ml. of 0.05 M Tris buffer, pH 8.0 (AIcohol 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 IO ml. of Tris buffer, pH 8.0 (Ammonium sulfate, Fraction 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 voIume 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- St. Louis. 1 (AS-1)). 4 Dried brewers' yeast, strain BSC, was kindly furnished by Anheuser-Busch, IIIC., 1028 ATP-METHIONINE REACTIONS 676 608 340 170 tion was cooled to 0" and 31 ml. of cold saturated ammonium sulfate were added. After another 5 minutes the precipitate waa removed by centrifu- gation for 10 minutea at 10,OOO X 9. 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 Trip buffer, pH 8.0 (Ammonium sulfate, Fraction 2 (AS-2)). unit; per m** P" m'. fits. protein 50.7 0.33 s.0 3.0 4.1 8.3 1.9 15.0 TABLB I Putijication oj Entyme Yeast extract (YE). ................... Alcohol fraction 2 (A-Z).. .............. Ammonium sulfate Fraction 1 (AS-1) . . " 2 (AS-2) . . 11 11 Concentn- tion of ensyme VdO 96f Wll. 16.9 15.2 34.0 28.4 TABLE 11 Requirenienls for Exchange of Parpar m*th ATP Components PIP incorporated into ATP I Complete. .............................. No ATP.. ............................ 'I methionine .......................... '6 MgClt.. ............................. I1 enzyme. ............................. rude 0.38 0.01 0.03 0.01 0.00 The conditions were the same aa those described for the assay of the ensymc. 50 7 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 w&s more stable, w8s used. Fraction AS-1 still contained some ATP-splitting activity but no detectable inorganic pyrophosphatase (17) or adenylic kinase (19). Requirements for PP-A TP Ezchnge-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 11). 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 waa 1 X lW4 M, and for half maximal rate it was 1 X M (Table 111). It might be pointed out that this procedure offers a relatively simple and rapid method for detecting and determining small amounts of L-methionine. SpecijiCity 05 L-Methionine Required-Of the naturally occurring amino acids, only methionine catalyzed a significant amount of exchange of PnPn and ATP with Fraction AS-1 (Table IV), Other amino acids alone or in various combinations gave values no higher than the control TABLB I11 Eflect of L-Melhionine Concentration on Rate of PP-ATP Ezchange Reaetion Concentration of bmcthionine x 104 Y 0 0.03 0.06 0.10 0.30 1 .o 3.0 7.0 15 PUP:' incorporated into ATP #mole 0.04 0.11 0.17 0.19 0.30 0.39 0.39 0.39 0.41 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-activat,ed exchange. The require- ment for methionine was found to be specific for the L form. D-Methionine wm 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 DL-ethionine, but, because extremely small amounts of methionine are ac- tive, the exchange found with ethionine may be due to contamination with methionine. Fomtation 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, Mg++, 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 Eflect of Varioua Amino Acids on PP-ATP Exchange Amino acid None. . . . . . . . . . . . . . . . . . . , . . . L-Methionine . . . . . . . . . . , . . . . L-Trypt ophan . . . . . . . . . . . . . . DL-Alanine . . , . . . . . . . . . . . . . , histidine. . . . . . . . . . . ~. . . . . L-Glutamic acid. . . . . . . . . . . . L-Isoleucine . . . . . . . . . . . . . . . . DL-Serine. . . . . . . . . . . . . . . . . . . L-Phenylalanine. . . , . . . . . . . . L-Valine . . . . . . . . . . . . . . . . . . . . L-Threonine . . . . . . . . . . . . . . . . L-Leucine . . . . . . . . . . . . . . , . . . L-Proline . . . . . . , . . . . . . . . . . . . DL-Homocysteine . . . . . . . . . . . Glycine. . . . . . . . . . . . . . . . . . . . . L-Tyrosine. . . . . . . . . . . . . . . . . . None. . . . . . . . . . , . . , . . . . . . . . L-Methionine . . . . . . . . . . . . . . D-Met hionine . . . . . . . . . . . . , . DL-Ethionine. . . . . . . . . . , . , . .............. (6 1 x 1WaM 1 x 10-3 " 1 x lo-' " 1 x lo-* " 1 x 10-3 " 1 x 10-3 '< 1 x lo-' " 1 x lo-' '( 1 x 10-a " I x lo-' " 1 x 10-2 " 1 x lo-' " 1 x lo-' " 1 x lo-' 1 x 10-3 '' 1.5 x M 1 x 10-3 6' 3 x 10-3 " 3 x 10-3 " Pa3PS* incorporated into ATP r~lr 0.01 0.13 0.01 0.01 0.02 0.01 0.01 0.00 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.02 0.04 0.41 0.05 0.04 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. When the enzyme, ATP, Mg++, 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 mp. The reason for this is not clear. P. BERG 1031 Experiment No. Nature of Reaction of ATP and Methionine-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 Pa2PE (Reactions 4 and 5). In the second, the products formed are adenyl me- Time TABLE V Enzymatic Formation of Methionine Hydrozanrate, A6P, and PP from ATP, Methionine, and H ydroz ylamine The values are given in micromoles per ml. I Met h io - nine hy- A* droxamatc 1 2 min. 30 60 30 60 30 30 30 Additions Complete No methionine Complete No methionine " ATP (4 44 I6 -- ASP - 1.79 0.95 0.16 __ A* +I .63 +0.79 PP - 1.47 2.17 0.14 0.27 1.81 0.09 0.09 +1.33 +1 .$MI 4-1.72 +o.oo 1.24 2.00 0.05 0.07 0.07 0.09 0.09 +1.19 4-1.93 +0.88 0.00 * 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 MgCl2,O.OIl M ATP, 0.01 M L-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 tls previously described, and PP was determined after conversion to inorganic phosphate with in- organic pyrophosphatase. Experiment 2. The reaction mixture (1 .I5 ml.) con- tained 0.09 M Tris buffer, pH 8.0,0.004 M MgClt, 0.011 M ATPJ* containing 6400 c.p.m. per pmole, 0.01 M L-methionine, 2.2 M hydroxylnmine, and I .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 WBS calculated from the PJ' in the nucleo- tide-free supernatant fluid. Separate aliquots were chromatographed on Dowex 1 to separate the A5P, and the total amount of A5P was determined from the op- tical density at 260 m, thionine and free PP (Reaction 6), which by reversal of the reaction con- verts P32Pa2 to ATP3?. (4) ATP + L-methionine e L-methionine-PP + A5P (5) L-Methionine-PP + Ps*Pa* e L-methionine-PJZP** + PP (6 ) ATP + t-methionine adenyl t-methionine + PP To distinguish between these two possibilities, the exchange of ASP-Cld to32 -,iTP-METHIONINE REACTIONS Labeled substrate and A'I'Y was studied. By inechanism (I), A5Y-CY4 should exchange with ATP at least as rapidly as does PS2P3?, and this should require L-methionine. By mechanism (2), there should be no exchange. It was found (Table VI) ! I Pm incorporated into PP i ]P~zincorpornted into ATP' -I 'A Time I A i-- TABLE VI Ezchange of A6P-CI4 and ATP - ! I ATP o ASP' Specific activity of compound isolated I Components I____ -~ _-- ~ ---- fflin. AR PP*SPa' 15 30 30 ps2pa2 15 c.6.m. per molr 1 c.p.m. per rrvrolr ~ Complete.. ................ j 15,000 < 100 < 100 No methionine. ............. 15,500 enzyme.. .............. i 15,000