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Preparation and properties of amino acid-adenylic acid mixed anhydrides
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
77,
13-19
(1958)
Preparation and Properties of Amino Acid-Adenylic Acid Mixed Anhydrides’ D. J. McCorquodale’ and Gerald C. Mueller Prom the McArdle
Memorial Laboratory, of Wisconsin, Madison,
Medical
School,
University
Wisconsin
Received January 28, 1958
INTRODUCTION Recent studies have indicated that one of the “activated” forms into proteins through which amino acids pass for their incorporation
involves a mixed anhydride of the amino acid with a form of adenylic acid (1). DeMoss et al. (2) and Wieland el al. (3) have provided direct, supporting evidence for this proposition. The initial step in amino acid activation may be formulated as follows:3 E + AA + ATP = E(AA-AMP)
+ PP
One difficulty in studying this system has been the lack method for synthesis of the anhydrides. The present paper convenient method which gives yields of the final purified hydride of 4&50%. Some properties of these anhydrides liver amino acid-activation system are reported.
(1)
of a good presents a mixed anin the rat
EXPERIMENTAL AMP and ATP were obtained from Pabst Laboratories Inc. DCC was prepared by the method of Schmidt et al. (4) from N,N’-dicyclohexylthiourea prepared according to Skita and Rolfes (5). Benzylmercaptoformyl chloride and the BMF 1 This work was supported by a grant from the Alexander and Margaret Stewart Trust Fund; Grant No. 1897-C5 from the U. S. Public Health Service; and an institutional grant from the American Cancer Society. 2 A postdoctorate fellow of the American Cancer Society. 3 The following abbreviations have been used: ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine 5’-phosphate; PC, phosphocreatine; AA, amino acid; E, amino acid-activating enzyme; DCC, dicyclohexylcarbodiimide; BMF, benzylmercaptoformyl; PP, pyrophosphate; RNA, ribonucleic acid. 13
MCCORQUODALE AND MUELLER
14
derivatives of glycine and nn-leucine were prepared according to Kollonitsch et al. (6). The amino acid-activating enzymes were prepared from rat liver according to Hoagland et al. (1). The washed “pH 5 enzyme” was finally dissolved in 0.05 M phosphate buffer, pH 6.5, to give a final protein concentration of about 5 mg./ml. AA-AMP was estimated by the hydroxamate method of Lipmann and Tuttle (7) as modified by DeMoss et al. (2). The formation of ATP from P32-P32 and AA-AMP by the reversal of Eq. (1) was followed by adsorbing the ATP-P32 on charcoal, hydrolyzing the terminal phosphates with 1 N HCl, and counting an aliquot of the supernatant according to DeMoss and Novelli’s (8) modification of the method of Crane and Lipmann (9). P32-activity was assayed using a dip counter. Identification of the adsorbed material as ATP-Pa2 was verified by elution of the adsorbed ATP-Ps2 from a 2 X 0.7 cm. Darco G-60 column which had been washed free of ortho- and pyrophosphate with 0.05 M sodium acetate buffer, pH 4.5, followed by electrophoresis of the eluate. The elution was effected with 10 ml. of 20% pyridine. The eluate was lyophilized and the residue dissolved in a small volume and subjected to high-potential paper electrophoresi@ (140 V./cm., 20 ma.) for 45 min. using acetic acid-formic acid buffer (pH 2.6). Under these conditions AMP, ADP, ATP, and PP are separated completely. The following methods were used for quantitative assay of functional groups in electrophoretically pure samples of the anhydrides: total phosphate by Fiske and SubbaRow (lo), anhydride linkages by the hydroxamate method of DeMoss et al. (2)) amino acid by C%pecific activity measurements, and adenine by hydrolysis of the anhydride and measurement of the optical density at 260 rnp. The hydroxamic acid formed from hydroxylamine and glycyl adenylate was identified by paper chromatography using the technique and solvent system of Hoagland et al. (1). The sample was compared to a pure preparation of glycine hydroxamic acid obtained by reacting salt-free hydroxylamine, prepared according to Houben (II), with free ethyl glycinate in ethanolic solution obtained by precipitating the chloride of ethyl glycinate hydrochloride with sodium ethylate in anhydrous ethanol. After standing overnight at -lO”C., the glycine hydroxamic acid separated out melting at 140°C. (dec.). After three recrystallizations from dilute methanol, the product gave a single, homogeneous, FeCla-sensitive spot on the chromatogram. Labeled pyrophosphate was prepared according to Lowenstein (12).
RESULTS
Synthesis of Glycyl-AMP
and m-Leucyl-AMP
Thirty milligrams of AMP and 90 mg. BMF-glycine (or 113 mg. BMF-nL-leucine) were dissolved in a mixture of 4.2 ml. pyridine and 0.45 ml. water at room temperature. Then 1.8 g. DCC was added, and the reaction mixture was cooled to -10°C. After 60 min. at -lO”C., * The high-voltage oration with Gilson
electrophoresis apparatus was designed and built Medical Electronics, Middleton, Wisconsin.
in collab-
AMINO
ACID-ADENYLIC
ACID
MIXED
ANHYDRIDES
15
during which time the reaction mixture was continually stirred with the aid of a magnetic stirrer, 30 ml. of cold ether was added, and the precipitate, containing the BMF-AA-AMP, was collected by sedimentation at 0°C. The precipitate was then washed several times with ether at 0°C. to remove residual pyridine and finally dried in a vacuum desiccator for about 20-30 min. The dry precipitate was then extracted with one IO-ml. and two 5-ml. portions of 0.01 N HCl, and any insoluble material was centrifuged off and discarded. To the clear HCl extract containing the BMF-AA-AMP was added 9 ml. of an aqueous solution of perbenzoic acid (11 mg./ml.). The oxidation was carried out for 20 min. at O”C., and the mixture was then extracted once with cold chloroform to remove excess perbenzoic acid. The aqueous phase, containing the AA-AMP, was then lyophilized. The residue from this lyophilization represents the crude product and may be stored for short periods in the cold. Further purification of the AA-AMP was carried out by passage through a Dowex 1 (chloride) column as described by DeMoss et al. (2). At pH 4.8, both glycyl-AMP and uL-leucyl-AkIt-’ passed through the column while free AMP was retained. The eluate containing the relnt’ively pure AA-AMP may be used directly or lyophilized and stored for short periods. The yield of this purified product was about 40-50 % of t.he theoretical based on adenylic acid used. This product’ contained kaces of free AMP and some free glycine or nL-leucinc. Since these contaminants did not interfere with the enzyme reactions, in most cases this product was routinely used without further purification for enzymic studies. However, in cases where more pure AA-AMP was desired, the eluate was lyophilized and purified by high-voltage electrophoresis. The synthesis can be followed conveniently with the use of a highpotential electrophoresis apparatus (150 V./cm., 20 ma.). Electrophoresis at pH 2.6 of the BMF-glycine-AMP-DCC reaction mixture (before removal of the BMF-group) revealed no bands moving to the cathode, while three ultraviolet-absorbing bands were present which migrated to the anode. These three bands were better resolved at pH 5.0 (0.025 M sodium acetate buffer) and consisted of free BMF-glycine, the fastest moving band; free AMP, intermediate in its migration velocity; and the BMF-glycyl-AMP, the slowest moving band. The yield of the BMF-glycyl-AMP is about 90% of theory based on optical density measurements at 260 rnp. Electrophoresis at, pH 2.6 of the
16
MCCORQUODALE
AND
MUELLER
reaction mixture after removal of the BMF-group revealed two bands migrating toward the cathode and one band moving slowly to the anode. The faster band, moving toward the cathode, is free glycine, which is non-ultraviolet-absorbing but ninhydrin-positive, while the slower band is the glycyl-AMP, which is ultraviolet-absorbing and ninhydrinpositive. The ultraviolet-absorbing band moving slowly toward the anode is free AMP. With the appearance of the glycyl-AMP band, there is a corresponding disappearance of the BMF-glycyl-AMP band. When the synthesis is carried out using BMF-glycine-2-C14, both the BMFglycyl-AMP and the glycyl-AMP bands are highly radioactive. Characterization of Glycyl-AMP The proposed structure of the AA-AMP
compounds is shown below.
NH2
c=o IP
R--&H I
HO-P-
NH2
0-CHe
-1 0 amino acid-adenylic
acid mixed anhydride
The following evidence supports this structure. (a) By analysis, the ratio of adenine-total phosphate-anhydride linkage-glycine is 1.0: 1.0: 1.05:0.99. (b) The ultraviolet spectrum of the material at pH 6.5 is identical with that of AMP. The 6-amino group is therefore not substituted since such substitutions shift the absorption band to longer wavelengths (13). (c) When the compound is treated with hydroxylamine in neutral solution, it reacts rapidly to yield one mole of glycine hydroxamate for each mole of adenine present. This indicates that the carboxyl group of the amino acid participates in an anhydride linkage in the compound. (d) The compound gives a red-violet ninhydrin reaction. (e) When the compound is subjected to high-potential electrophoresis at neutral pH, the compound does not move from the origin, while at acid pH (2.6) it migrates to the cathode. These electrophoretic migration characteristics are consistent with the formulation shown. (f) The compound is rapidly hydrolyzed above pH 7.0 but is reasonably stable
AMINO
ACID-ADENYLIC
ACID
MIXED
1'3
ANHYDRIDES
between pH 2.0 and 7.0. (g) Electrophoresis of the mild, alkaline drolysis products of the pure compound at pH 2.6 showed only bands. One, moving slowly to the anode, was ultraviolet-absorbing indistinguishable from AMP. The other, moving to the cathode, non-ultraviolet-absorbing but ninhydrin-positive and was identified free glycine.
hytwo and was as
Enzymic Formation of ATP Since AA-AMP anhydrides are unstable at alkaline pH, it was necessary to incubate them in a system operating at acid pH. It has been found that the 105,000 X g supernatant from homogenates of normal rat liver prepared according to Zamecnik and Keller (14), together with active microsomes, catalyzes the incorporation of amino acids into protein at pH 6.5. Thus, the enzymic formation of ATP was demonstrated at pH 6.5 using the amino acid-activating enzymes obtained from this 105,000 X g supernatant. The data showing the ATP formed by the reversal of Eq. (1) are given in Table I. For identification of the adsorbed material, the charcoal was eluted, and the eluate was lyophilized and subjected to high-potential electrophoresis. The electrophoregram showed a highly radioactive, ulTABLE I Formation of ATP from Glycyl-AMP and m-Leucyl-AMP The complete system consisted of 65 pmoles phosphate buffer, pH 6.5, 2 pmoles 5.2 pmoles glycyl-AMP or 5.0 MgClz , 10 pmoles PP32 (6075 counts/min./pmole), pmoles m-leucyl-AMP, 0.5 ml. amino acid-activating enzyme (2.3 mg. protein) in a final volume of 1.3 ml. Incubation was carried out at 37°C. for 8 min. at pH 6.5 under an atmosphere of air. Anhydride
tested
Component omitted
ATP formed p??Wk
Glycyl-AMP
nn-Leucyl-AMP
None Glycyl-AMP Glycyl-AMP0 Enzyme None nn-Leucyl-AMP m-Leucyl-AMPa Enzyme
0.40 0.00
0.01 0.00 0.19 0.00 0.01 0.00
Q In these tubes, 5 pmoles AMP and 5 rmoles of either glycine or Dr.-leucine were substituted for the glycyl-AMP or nn-leucyl-AMP, respectively.
18
MCCORQUODALE
AND
MUELLER
traviolet-absorbing band with migration characteristics indistinguishable from a known sample of ATP. The ultraviolet spectrum of this band was identical with that of ATP. Since it is now clear that AA-AMP type compounds participate in Eq. (l), attempts were made to isolate any anhydride accumulated during incubation. Incubations were carried out at pH 6.5 for 5-10 min. with glycine-2-Cl4 (2 pmoles) or nn-leucine-l-Cl4 (2 pmoles), ATP (5 pmoles), Mg (2 pmoles), PC (20 pmoles), creatine kinase (6 pg.), and the 0.7 ml. of 105,000 X g supernatant from 10 % rat liver homogenates. The synthetic AA-AMP was then added as a cool pool at the beginning, at the end, or at 1-min. intervals throughout the incubation, and reisolated by high-potential electrophoresis immediately after incubation was complete. If unbound AA-AMP-C4 was formed in the ensymic reaction, the reisolated AA-AMP should have contained Cl4 activity. However, no radioactivity was detected in the reisolated anhydride. DISCUSSION
DeMoss et al. (2) have reported that attempts to synthesize n-leucylAMP by acylating AMP with acetyl-, carboethoxy-, or carbobenzoxyleucine were unsuccessful since these protecting groups could not be removed from the N-acylated-AA-AMP without concomitant breakage of the anhydride linkage. The report by Kollonitsch et al. (6) that the BMF group could be removed from BMF-amino acids under extremely mild oxidizing conditions, in contrast to the relatively severe reducing conditions required to remove the acetyl-, carboethoxy-, or carbobenzoxy groups, initiated this work. The success in the present synthesis demonstrates the extreme ease with which the BMF group can be removed from even a relatively unstable compound and indicates the potential versatility of the BMF-protecting group. Berg (15) has recently reported another method of mixed-anhydride synthesis which gives yields of the same order as reported here. His synthetic AA-AMP compounds will also form ATP when incubated with PP and an amino acid-activating enzyme preparation from yeast or Escherichia coli. It has been previously shown by Wieland et al. (3) that nn-valyl-AMP and PP will form ATP when incubated with the amino acid-activating enzymes from rat liver and that n-valine stimulates the rate of PP-ATP exchange in this system (1). n-Leucine also stimulates the PP-ATP exchange and will form its corresponding hydroxamic acid, as will glycine, when incubated in this same system with hydroxylamine. The
AMINO
ACID-ADENYLIC
IICID
MIXED
ANHYDRIDES
19
present results, which demonstrate that nn-leucyl-AMP and glycylAMP form ATP in this system, provide further evidence for the involvement of AA-AMP type compounds in the incorporation of amino acids into proteins. Support is therefore given to the formulation of the reaction given in Eq. (1). It, is interesting to note that the amino acid-activating enzyme preparation formed as much ATP from glycyl-AMP as from an equivalent amount of nn-leucyl-AMP. From Table I, 0.19 pmole ATI’ is formed from 5.0 pmoles of nn-leucyl-AMP or, considering that only the L-form is utilized (a), from 2.5 pmoles L-leucyl-AMP. Similarly, 0.40 pmole ATP is formed from 5.2 pmoles glycyl-AMP or 0.19 pmole per 2.5 pmoles anhydride. Glycine does not enhance the PP-ATP exchange in E. coli extracts, while n-leucine does (8). DeMoss et al. (2) and Berg (15) have also reported that adenyl derivatives of those amino acids which do not themselves significantly promote the PI’-ATP exchange will form ATP in the presence of PP and the activat,ing enzyme. Since attempts to isolate AA-AMP from the amino acid activation system were unsuccessful, it is probable that at no time does the AAAMP exist in a free state but is always bound either to the enzyme surface or to the RNA fragments (16, 17) to which it, is transferred without passing through a free form. REFERENCES 1. HOAGLAND, h’I. B., KELLER, E. B., AND ZAMECNIK, I’. C., .I. Biol. Chem. 218, 345 (1956). 2. DEMOSS, J. A., GENUTH, S. M., ASD NOVELLI, G. D., Proc. Natl. Acad. Sci. U. S. 42, 325 (1956). 3. WIELAND, R., NIEMANN, E., AND PFLEIDERER, G., Angew. Chem. 68,305 (1956). 4. SCHMIDT, E., HITZLER, F., AND LAHDE, E., Ber. ‘71, 1933 (1938). 5. SKITA, A., AND ROLFES, H., Ber. 63, 1247 (1920). 6. KOLLONITSCH, J., GABOR, V., AND HAJOS, A., Chem. Ber. 89, 2293 (1956). 7. LIP~MANN, F., AND TUTTLE, L. C., J. Biol. Chem. 169, 21 (1945). 8. DEMOSS, J. A., AND NOVELLI, G. D., Biochim. et Biophys. Acta 22, 49 (1957). 9. CRANE, R. K., AND LIPMANN, F., J. Biol. Chem. 201, 235 (1953). 10. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 11. HOUBEN, J., J. prakt. Chem. 106, 7 (1922). 12. LOWENSTEIN, J. M., Biochem. J. 66, 197 (1957). 13. KOSSEL, A., 2. physiol. Chem. 12, 241 (1888). 14. ZAMECNIK, P. C., AND KELLER, E. B., J. Biol. Chem. 209, 337 (1954). 15. BERG, P., Federatio?z Proc. 16, 152 (1957). 16. HOAGLAND, M. B., AND ZAMECNIK, P. C., Federation Proc. 16, 197 (1957). 17. HObGLANI), X. B., ZAMECNIK, P. C., ASD STEPHENSON, M. L., Biochim. et Biophys. Acta 24, 215 (1957).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
77,
13-19
(1958)
Preparation and Properties of Amino Acid-Adenylic Acid Mixed Anhydrides’ D. J. McCorquodale’ and Gerald C. Mueller Prom the McArdle
Memorial Laboratory, of Wisconsin, Madison,
Medical
School,
University
Wisconsin
Received January 28, 1958
INTRODUCTION Recent studies have indicated that one of the “activated” forms into proteins through which amino acids pass for their incorporation
involves a mixed anhydride of the amino acid with a form of adenylic acid (1). DeMoss et al. (2) and Wieland el al. (3) have provided direct, supporting evidence for this proposition. The initial step in amino acid activation may be formulated as follows:3 E + AA + ATP = E(AA-AMP)
+ PP
One difficulty in studying this system has been the lack method for synthesis of the anhydrides. The present paper convenient method which gives yields of the final purified hydride of 4&50%. Some properties of these anhydrides liver amino acid-activation system are reported.
(1)
of a good presents a mixed anin the rat
EXPERIMENTAL AMP and ATP were obtained from Pabst Laboratories Inc. DCC was prepared by the method of Schmidt et al. (4) from N,N’-dicyclohexylthiourea prepared according to Skita and Rolfes (5). Benzylmercaptoformyl chloride and the BMF 1 This work was supported by a grant from the Alexander and Margaret Stewart Trust Fund; Grant No. 1897-C5 from the U. S. Public Health Service; and an institutional grant from the American Cancer Society. 2 A postdoctorate fellow of the American Cancer Society. 3 The following abbreviations have been used: ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine 5’-phosphate; PC, phosphocreatine; AA, amino acid; E, amino acid-activating enzyme; DCC, dicyclohexylcarbodiimide; BMF, benzylmercaptoformyl; PP, pyrophosphate; RNA, ribonucleic acid. 13
MCCORQUODALE AND MUELLER
14
derivatives of glycine and nn-leucine were prepared according to Kollonitsch et al. (6). The amino acid-activating enzymes were prepared from rat liver according to Hoagland et al. (1). The washed “pH 5 enzyme” was finally dissolved in 0.05 M phosphate buffer, pH 6.5, to give a final protein concentration of about 5 mg./ml. AA-AMP was estimated by the hydroxamate method of Lipmann and Tuttle (7) as modified by DeMoss et al. (2). The formation of ATP from P32-P32 and AA-AMP by the reversal of Eq. (1) was followed by adsorbing the ATP-P32 on charcoal, hydrolyzing the terminal phosphates with 1 N HCl, and counting an aliquot of the supernatant according to DeMoss and Novelli’s (8) modification of the method of Crane and Lipmann (9). P32-activity was assayed using a dip counter. Identification of the adsorbed material as ATP-Pa2 was verified by elution of the adsorbed ATP-Ps2 from a 2 X 0.7 cm. Darco G-60 column which had been washed free of ortho- and pyrophosphate with 0.05 M sodium acetate buffer, pH 4.5, followed by electrophoresis of the eluate. The elution was effected with 10 ml. of 20% pyridine. The eluate was lyophilized and the residue dissolved in a small volume and subjected to high-potential paper electrophoresi@ (140 V./cm., 20 ma.) for 45 min. using acetic acid-formic acid buffer (pH 2.6). Under these conditions AMP, ADP, ATP, and PP are separated completely. The following methods were used for quantitative assay of functional groups in electrophoretically pure samples of the anhydrides: total phosphate by Fiske and SubbaRow (lo), anhydride linkages by the hydroxamate method of DeMoss et al. (2)) amino acid by C%pecific activity measurements, and adenine by hydrolysis of the anhydride and measurement of the optical density at 260 rnp. The hydroxamic acid formed from hydroxylamine and glycyl adenylate was identified by paper chromatography using the technique and solvent system of Hoagland et al. (1). The sample was compared to a pure preparation of glycine hydroxamic acid obtained by reacting salt-free hydroxylamine, prepared according to Houben (II), with free ethyl glycinate in ethanolic solution obtained by precipitating the chloride of ethyl glycinate hydrochloride with sodium ethylate in anhydrous ethanol. After standing overnight at -lO”C., the glycine hydroxamic acid separated out melting at 140°C. (dec.). After three recrystallizations from dilute methanol, the product gave a single, homogeneous, FeCla-sensitive spot on the chromatogram. Labeled pyrophosphate was prepared according to Lowenstein (12).
RESULTS
Synthesis of Glycyl-AMP
and m-Leucyl-AMP
Thirty milligrams of AMP and 90 mg. BMF-glycine (or 113 mg. BMF-nL-leucine) were dissolved in a mixture of 4.2 ml. pyridine and 0.45 ml. water at room temperature. Then 1.8 g. DCC was added, and the reaction mixture was cooled to -10°C. After 60 min. at -lO”C., * The high-voltage oration with Gilson
electrophoresis apparatus was designed and built Medical Electronics, Middleton, Wisconsin.
in collab-
AMINO
ACID-ADENYLIC
ACID
MIXED
ANHYDRIDES
15
during which time the reaction mixture was continually stirred with the aid of a magnetic stirrer, 30 ml. of cold ether was added, and the precipitate, containing the BMF-AA-AMP, was collected by sedimentation at 0°C. The precipitate was then washed several times with ether at 0°C. to remove residual pyridine and finally dried in a vacuum desiccator for about 20-30 min. The dry precipitate was then extracted with one IO-ml. and two 5-ml. portions of 0.01 N HCl, and any insoluble material was centrifuged off and discarded. To the clear HCl extract containing the BMF-AA-AMP was added 9 ml. of an aqueous solution of perbenzoic acid (11 mg./ml.). The oxidation was carried out for 20 min. at O”C., and the mixture was then extracted once with cold chloroform to remove excess perbenzoic acid. The aqueous phase, containing the AA-AMP, was then lyophilized. The residue from this lyophilization represents the crude product and may be stored for short periods in the cold. Further purification of the AA-AMP was carried out by passage through a Dowex 1 (chloride) column as described by DeMoss et al. (2). At pH 4.8, both glycyl-AMP and uL-leucyl-AkIt-’ passed through the column while free AMP was retained. The eluate containing the relnt’ively pure AA-AMP may be used directly or lyophilized and stored for short periods. The yield of this purified product was about 40-50 % of t.he theoretical based on adenylic acid used. This product’ contained kaces of free AMP and some free glycine or nL-leucinc. Since these contaminants did not interfere with the enzyme reactions, in most cases this product was routinely used without further purification for enzymic studies. However, in cases where more pure AA-AMP was desired, the eluate was lyophilized and purified by high-voltage electrophoresis. The synthesis can be followed conveniently with the use of a highpotential electrophoresis apparatus (150 V./cm., 20 ma.). Electrophoresis at pH 2.6 of the BMF-glycine-AMP-DCC reaction mixture (before removal of the BMF-group) revealed no bands moving to the cathode, while three ultraviolet-absorbing bands were present which migrated to the anode. These three bands were better resolved at pH 5.0 (0.025 M sodium acetate buffer) and consisted of free BMF-glycine, the fastest moving band; free AMP, intermediate in its migration velocity; and the BMF-glycyl-AMP, the slowest moving band. The yield of the BMF-glycyl-AMP is about 90% of theory based on optical density measurements at 260 rnp. Electrophoresis at, pH 2.6 of the
16
MCCORQUODALE
AND
MUELLER
reaction mixture after removal of the BMF-group revealed two bands migrating toward the cathode and one band moving slowly to the anode. The faster band, moving toward the cathode, is free glycine, which is non-ultraviolet-absorbing but ninhydrin-positive, while the slower band is the glycyl-AMP, which is ultraviolet-absorbing and ninhydrinpositive. The ultraviolet-absorbing band moving slowly toward the anode is free AMP. With the appearance of the glycyl-AMP band, there is a corresponding disappearance of the BMF-glycyl-AMP band. When the synthesis is carried out using BMF-glycine-2-C14, both the BMFglycyl-AMP and the glycyl-AMP bands are highly radioactive. Characterization of Glycyl-AMP The proposed structure of the AA-AMP
compounds is shown below.
NH2
c=o IP
R--&H I
HO-P-
NH2
0-CHe
-1 0 amino acid-adenylic
acid mixed anhydride
The following evidence supports this structure. (a) By analysis, the ratio of adenine-total phosphate-anhydride linkage-glycine is 1.0: 1.0: 1.05:0.99. (b) The ultraviolet spectrum of the material at pH 6.5 is identical with that of AMP. The 6-amino group is therefore not substituted since such substitutions shift the absorption band to longer wavelengths (13). (c) When the compound is treated with hydroxylamine in neutral solution, it reacts rapidly to yield one mole of glycine hydroxamate for each mole of adenine present. This indicates that the carboxyl group of the amino acid participates in an anhydride linkage in the compound. (d) The compound gives a red-violet ninhydrin reaction. (e) When the compound is subjected to high-potential electrophoresis at neutral pH, the compound does not move from the origin, while at acid pH (2.6) it migrates to the cathode. These electrophoretic migration characteristics are consistent with the formulation shown. (f) The compound is rapidly hydrolyzed above pH 7.0 but is reasonably stable
AMINO
ACID-ADENYLIC
ACID
MIXED
1'3
ANHYDRIDES
between pH 2.0 and 7.0. (g) Electrophoresis of the mild, alkaline drolysis products of the pure compound at pH 2.6 showed only bands. One, moving slowly to the anode, was ultraviolet-absorbing indistinguishable from AMP. The other, moving to the cathode, non-ultraviolet-absorbing but ninhydrin-positive and was identified free glycine.
hytwo and was as
Enzymic Formation of ATP Since AA-AMP anhydrides are unstable at alkaline pH, it was necessary to incubate them in a system operating at acid pH. It has been found that the 105,000 X g supernatant from homogenates of normal rat liver prepared according to Zamecnik and Keller (14), together with active microsomes, catalyzes the incorporation of amino acids into protein at pH 6.5. Thus, the enzymic formation of ATP was demonstrated at pH 6.5 using the amino acid-activating enzymes obtained from this 105,000 X g supernatant. The data showing the ATP formed by the reversal of Eq. (1) are given in Table I. For identification of the adsorbed material, the charcoal was eluted, and the eluate was lyophilized and subjected to high-potential electrophoresis. The electrophoregram showed a highly radioactive, ulTABLE I Formation of ATP from Glycyl-AMP and m-Leucyl-AMP The complete system consisted of 65 pmoles phosphate buffer, pH 6.5, 2 pmoles 5.2 pmoles glycyl-AMP or 5.0 MgClz , 10 pmoles PP32 (6075 counts/min./pmole), pmoles m-leucyl-AMP, 0.5 ml. amino acid-activating enzyme (2.3 mg. protein) in a final volume of 1.3 ml. Incubation was carried out at 37°C. for 8 min. at pH 6.5 under an atmosphere of air. Anhydride
tested
Component omitted
ATP formed p??Wk
Glycyl-AMP
nn-Leucyl-AMP
None Glycyl-AMP Glycyl-AMP0 Enzyme None nn-Leucyl-AMP m-Leucyl-AMPa Enzyme
0.40 0.00
0.01 0.00 0.19 0.00 0.01 0.00
Q In these tubes, 5 pmoles AMP and 5 rmoles of either glycine or Dr.-leucine were substituted for the glycyl-AMP or nn-leucyl-AMP, respectively.
18
MCCORQUODALE
AND
MUELLER
traviolet-absorbing band with migration characteristics indistinguishable from a known sample of ATP. The ultraviolet spectrum of this band was identical with that of ATP. Since it is now clear that AA-AMP type compounds participate in Eq. (l), attempts were made to isolate any anhydride accumulated during incubation. Incubations were carried out at pH 6.5 for 5-10 min. with glycine-2-Cl4 (2 pmoles) or nn-leucine-l-Cl4 (2 pmoles), ATP (5 pmoles), Mg (2 pmoles), PC (20 pmoles), creatine kinase (6 pg.), and the 0.7 ml. of 105,000 X g supernatant from 10 % rat liver homogenates. The synthetic AA-AMP was then added as a cool pool at the beginning, at the end, or at 1-min. intervals throughout the incubation, and reisolated by high-potential electrophoresis immediately after incubation was complete. If unbound AA-AMP-C4 was formed in the ensymic reaction, the reisolated AA-AMP should have contained Cl4 activity. However, no radioactivity was detected in the reisolated anhydride. DISCUSSION
DeMoss et al. (2) have reported that attempts to synthesize n-leucylAMP by acylating AMP with acetyl-, carboethoxy-, or carbobenzoxyleucine were unsuccessful since these protecting groups could not be removed from the N-acylated-AA-AMP without concomitant breakage of the anhydride linkage. The report by Kollonitsch et al. (6) that the BMF group could be removed from BMF-amino acids under extremely mild oxidizing conditions, in contrast to the relatively severe reducing conditions required to remove the acetyl-, carboethoxy-, or carbobenzoxy groups, initiated this work. The success in the present synthesis demonstrates the extreme ease with which the BMF group can be removed from even a relatively unstable compound and indicates the potential versatility of the BMF-protecting group. Berg (15) has recently reported another method of mixed-anhydride synthesis which gives yields of the same order as reported here. His synthetic AA-AMP compounds will also form ATP when incubated with PP and an amino acid-activating enzyme preparation from yeast or Escherichia coli. It has been previously shown by Wieland et al. (3) that nn-valyl-AMP and PP will form ATP when incubated with the amino acid-activating enzymes from rat liver and that n-valine stimulates the rate of PP-ATP exchange in this system (1). n-Leucine also stimulates the PP-ATP exchange and will form its corresponding hydroxamic acid, as will glycine, when incubated in this same system with hydroxylamine. The
AMINO
ACID-ADENYLIC
IICID
MIXED
ANHYDRIDES
19
present results, which demonstrate that nn-leucyl-AMP and glycylAMP form ATP in this system, provide further evidence for the involvement of AA-AMP type compounds in the incorporation of amino acids into proteins. Support is therefore given to the formulation of the reaction given in Eq. (1). It, is interesting to note that the amino acid-activating enzyme preparation formed as much ATP from glycyl-AMP as from an equivalent amount of nn-leucyl-AMP. From Table I, 0.19 pmole ATI’ is formed from 5.0 pmoles of nn-leucyl-AMP or, considering that only the L-form is utilized (a), from 2.5 pmoles L-leucyl-AMP. Similarly, 0.40 pmole ATP is formed from 5.2 pmoles glycyl-AMP or 0.19 pmole per 2.5 pmoles anhydride. Glycine does not enhance the PP-ATP exchange in E. coli extracts, while n-leucine does (8). DeMoss et al. (2) and Berg (15) have also reported that adenyl derivatives of those amino acids which do not themselves significantly promote the PI’-ATP exchange will form ATP in the presence of PP and the activat,ing enzyme. Since attempts to isolate AA-AMP from the amino acid activation system were unsuccessful, it is probable that at no time does the AAAMP exist in a free state but is always bound either to the enzyme surface or to the RNA fragments (16, 17) to which it, is transferred without passing through a free form. REFERENCES 1. HOAGLAND, h’I. B., KELLER, E. B., AND ZAMECNIK, I’. C., .I. Biol. Chem. 218, 345 (1956). 2. DEMOSS, J. A., GENUTH, S. M., ASD NOVELLI, G. D., Proc. Natl. Acad. Sci. U. S. 42, 325 (1956). 3. WIELAND, R., NIEMANN, E., AND PFLEIDERER, G., Angew. Chem. 68,305 (1956). 4. SCHMIDT, E., HITZLER, F., AND LAHDE, E., Ber. ‘71, 1933 (1938). 5. SKITA, A., AND ROLFES, H., Ber. 63, 1247 (1920). 6. KOLLONITSCH, J., GABOR, V., AND HAJOS, A., Chem. Ber. 89, 2293 (1956). 7. LIP~MANN, F., AND TUTTLE, L. C., J. Biol. Chem. 169, 21 (1945). 8. DEMOSS, J. A., AND NOVELLI, G. D., Biochim. et Biophys. Acta 22, 49 (1957). 9. CRANE, R. K., AND LIPMANN, F., J. Biol. Chem. 201, 235 (1953). 10. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 66, 375 (1925). 11. HOUBEN, J., J. prakt. Chem. 106, 7 (1922). 12. LOWENSTEIN, J. M., Biochem. J. 66, 197 (1957). 13. KOSSEL, A., 2. physiol. Chem. 12, 241 (1888). 14. ZAMECNIK, P. C., AND KELLER, E. B., J. Biol. Chem. 209, 337 (1954). 15. BERG, P., Federatio?z Proc. 16, 152 (1957). 16. HOAGLAND, M. B., AND ZAMECNIK, P. C., Federation Proc. 16, 197 (1957). 17. HObGLANI), X. B., ZAMECNIK, P. C., ASD STEPHENSON, M. L., Biochim. et Biophys. Acta 24, 215 (1957).