Some Problems Concerning the Activation of Amino Acids by G

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19 Th. Wieland, E. Niemann, and G. Pfleiderer, Angew. Chem., 68, 305, 1956. 20 F. H. Bergmann and P. Berg, unpublished. 21 E. J. Ofengand, and P. Berg, unpublished. 22 A. M. Crestfield, K. C. Smith, and F. W. Allen, J. Biol. Chem., 216, 185, 1955. 23 Z. Dische, E. Chargaff, and J. N. Davidson, (eds.), The Nucleic Acids, 1 (New York: Academic Press, 1955), 285. 24 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265, 1951. 5 We are grateful to Drs. Leon Heppel, Uriel Littauer, and Barry Commoner for some of these ribopolynucleotide preparations. 26 M. Grunberg-Manago, P. J. Ortiz, and S. Ochoa, Biochim. et Biophys. Acta, 20, 269, 1956. 27 R. W. Holley, J. Am. Chem. Soc., 79, 658, 1957. 28 A. L. Dounce, Enzymologia, 15, 251, 1952. 29 E. F. Gale, Enzymes: Units of Biological Structure and Function (New York: Academic Press, 1956), p. 49. 30 S. Spiegelman, Symposium on the Chemical Basis of Heredity (Baltimore: Johns Hopkins Press, 1957), p. 232. 31 F. Lipmann, Currents in Biochemical Research (New York: Interscience Publishers, Inc., 1956), p. 241. 32 G. Gamow, A. Rich, and M. Yeas, Advances in Biol. and Med. Phys., 4, 23, 1956. 33 M. B. Hoagland, P. C. Zamecnik, and M. L. Stephenson, Biochim. et Biophys. Acta, 24, 215, 1957. 34 S. Brenner, these PROCEEDINGS 43, 687, 1957. 3 J. R. Stern, J. Am. Chem. Soc., 77, 5194, 1955.

SOME PROBLEMS CONCERNING THE ACTIVATION OF AMINO ACIDS BY G. DAVID NOVELLI BIOLOGY DIVISION, OAK RIDGE NATIONAL LABORATORY* Any enzyme system that is proposed to account for the activation of amino acids prior to their conversion to proteins must have certain characteristics: (1) the reaction must involve all the naturally occurring amino acids; (2) there may rea- sonably be a correspondence between the degree of activation of a given amino acid and the frequency of occurrence of that amino acid in proteins; and (3) the proposed reaction should be present in all living cells. Hoagland' discovered an amino acid-dependent exchange between inorganic pyrophosphate (PP) and ATP in extracts of rat liver that appears to reflect a system for the carboxyl activation of amino acids. DeMoss and Novelli2 found this exchange reaction widely distributed in micro-organisms. Since the exchange reaction seemed to involve many of the naturally occurring amino acids and also be- cause the reaction was found in partially resolved systems that catalyze the incor- poration of amino acids into proteins, the amino acid-dependent PP-ATP exchange system has been implicated as the enzyme system responsible for the activation of amino acids for the biosynthesis of proteins.3-5 My purpose in this discussion is to report some observations on the activation of amino acids, as reflected by the PP-ATP exchange reaction, that are inconsistent with the idea that this system is responsible for the activation of amino acids for protein biosynthesis. These observations may be real or artifacts, but in either Downloaded by guest on October 4, 2021 VOL. 44, 1958 SYMPOSIUM 87

case they must be adequately explained if we are to evaluate the function of such systems in the biosynthesis of proteins. It has been indicated in previous papers in this symposium8' 7 that the activation of amino acids is generally measured by one of two methods: (1) the amino acid- dependent exchange of inorganic pyrophosphate with ATP: AA ATP + pp32 ATP32 + pp32 and (2) the formation of amino acid hydroxamates when preparations are incubated with amino acids, ATP, and relatively high concentrations of hydroxylamine: H H R-C-COOH + ATP + NH20H R-C-CONHOH + PP + AMP .I NH2 NH2 I should like to examine briefly the sort of information obtained through the ap- plication of both these methods to the general problem of the activation of amino acids and to make some attempt to interpret the various findings. The Amino Acid-dependent PP-A TP Exchange.-During studies on the activation of amino acids by crude extracts of Escherichia coli, DeMoss and Novelli2 observed that only eight to ten (cysteine, methionine, leucine, isoleucine, valine, tyrosine, tryptophan, phenylalanine, histidine, and possibly serine) of the naturally occurring amino acids significantly stimulated the exchange of PP with ATP. The word "stimulated" is used advisedly, since, in crude extracts of all tissues, some PP or- dinarily exchanges with ATP in the absence of added amino acids. Upon the addition of a complete mixture of amino acids or of certain individual amino acids, the rate of exchange of PP with ATP increases. This background exchange, in the absence of added amino acids, is troublesome in calculating the effect of individual amino acids. This endogenous exchange is caused partly, but not entirely, by the presence of amino acids in the crude extracts; it can be reduced but not completely eliminated by dialysis, by repeated reprecipitation of the protein with saturated ammonium sulfate, or bv treatment of the extracts with charcoal or with a mixture of anion and cation exchange resins. The observation that not all the amino acids stimulated the PP-ATP exchange reaction in the E. coli preparation can be variously interpreted. One may assume that (a) the enzymes required for the exchange with the apparently inactive amino acids were destroyed during the preparation of the extracts; (b) these enzymes were already saturated with their respective amino acid and therefore the further addition of amino acid could not elicit additional re- sponse (this amounts to saying that the endogenous exchange is being catalyzed by the inactive amino acids that are already saturating their respective enzymes); (c) it could be assumed that the inactive amino acids are activated through a mechanism not involving a PP-ATP exchange. In Table 1, data are presented that compare the stimulation of the PP-ATP exchange by individual amino acids in extracts of five micro-organisms, four animal tissues, and one plant tissue extract. In the extracts from the micro-organisms, rat liver, rat kidney, and peas (although there is some individual variation), all the Downloaded by guest on October 4, 2021 88 SYMPOSIUM PROC. N. A. S.

significantly active amino acids are among those mentioned previously. With extracts from guinea-pig tissues, on the other hand, there seems to be a small but significant exchange with almost all the amino acids. Although the exchange rates with mammalian tissues are only about one-tenth the rate in microbial extracts, the highest exchange rates with individual amino acids are seen largely with those amino acids that are active in micro-organisms. I think we can generalize these observations by saying that alanine, arginine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, proline, hydroxyproline, and lysine have little or no ability to catalyze the PP-ATP exchange reaction. In this connection it is interesting that the combination of asparagine, aspartic acid, glutamine, and glutamic acid repre- sents a large percentage of the total amino acid residues of a number of proteins (i.e., insulin, 26 per cent; egg albumin, 21 per cent; edestine, 28 per cent; and zein,

TABLE 1 RATE OF THE PP-ATP EXCHANGE WITH INDIVIDUAL AMINO ACIDS IN VARIOUS EXTRACTS* -PYROPHOSPHATE-ADENOSINE TRIPHOSPHATE EXCHANGE (JLMOLEs/HR/MG OF PROTEIN)- Guinea- Guinea- E. M. lacti- N. A. aero- A. fl8- Rat Rat Pig Pig Green AMINO ACID coli lyticus craesa genes cheri Liver Kidney Pancreas Liver Peas CompleteAA 7.30 6.40 1.67 21.0 12.4 0.37 0.23 1.20 0.99 0.85 Alanine 0 0 0.14 0.33 0.05 0 0 0.02 0.01 0 Arginine 0 0 0 0 0.12 0 0 0.05 0 0 Aspartic acid 0 0 0.14 0 0.19 ... 0.06 0 0 0 Asparagine 0 0 0.14 0 0.31 ... 0 0.11 0 0 Glutamic acid 0.07 0 0.14 0 0.37 0 0 0.02 0 0 Glutamine 0 0 0 0 0.37 0 0 0 0 0 Glycine 0 0 0 0 0.05 0 0 0.03 0.07 0 Hydroxy- proline 0 0.08 0 0 0 0 0 0 0.05 0 Lysine 0 0 0 0 0 0 0 0 0.05 0 Proline 0 0 0 0 0 0 0 0.08 0.03 0 Serine 0.33 0.41 0.36 0 0 0 0 0.29 0.05 0 Threonine 0 0 0.14 0 0 0 0 0.23 0.02 .0 Tryptophan 0.36 0.98 0.14 0 0.20 0.04 0 0.06 0.06 0.12 Histidine 0.57 0 0.44 0 0 0.02 0 0.25 0.43 0 Phenylalanine 0.95 0 0.55 0.33 0 0.02 0.13 0 0.13 0 Methionine 2.53 0.34 0 2.02 2.34 0.12 0.02 0 0.55 0.02 Tyrosine 3.00 0.41 0.87 2.37 0.12 0.13 0.04 0.25 0.19 0.29 Valine 3.00 2.07 0.65 9.14 3.60 0.04 0 0.08 0.32 0.27 Leucine 3.46 3.34 1.59 10.04 5.02 0.15 0.37 0.20 0.30 0.30 Isoleucine 5.12 5.01 0.16 16.59 2.34 0.10 0.22 0.08 0.71 0.04 Cysteine 1.59 0.55 4.73 0.12 0.28 0.15 0.06 0.02 * Data on E. coli, Micrococcus lactilyticus, Neurospora crassa, and Aerobacter aerogenes: J. A. DeMoss, Ph.D. thesis, Dept. of Microbiology, Western Reserve University School of Medicine, 1957. Data on A. fischeri: M. J. Cormier and G. D. Novelli, unpublished observations. Data on peas: J. W. Davis and G. D. Novelli, Arch. Biochem. and Biophys. (in press). 29 per cent).8 If it should prove that these amino acids are not activated by a process involving a PP-ATP exchange, then some other means of activating these amino acids needs to be found if the activating system under discussion is to be implicated in protein synthesis. DeMoss and Novelli2' 4have suggested that the lat- ter group of amino acids might be activated by an anhydride exchange reaction, but evidence in support of this suggestion has not been obtained. The Formation of Amino Acid Hydroxamates.-It has been mentioned that amino acid carboxyl activation can also be measured through the formation of hydroxamic acid. This method has been used for the isolation and purification of several amino acid-activating enzymes.9-12 In the hope of learning something about a possible activation of the "inactive" amino acids, we compared the rate of the PP-ATP ex- Downloaded by guest on October 4, 2021 VOL. 44) 1958 SYMPOSIUM 89

TABLE 2 COMPARISON OF RATE OF PP-ATP EXCHANGE WITH HYDROXAMATE IN Elcherichia coli* RATE RATE (MAMOLEs/HR/MG (pMOLEs/HR/MG OF PROTEIN) OF PROTEIN) Ex- Hydrox- EXCHANGSE Ex- Hydrox- EXCHANGE AMINO ACID change amate HYDROXAMATE AMINO ACID change amate HYDROXAMATE

Alanine 0 0.008 .. Serine 0.33 0.011 30

Arginine 0 0.019 .. Threonine 0 0.013

Aspartic acid 0 0.447 .. Tryptophan 0.41 0.025 16 Asparagine 0 0.332 Histidine 0.62 0.002 310 Glutamic acid 0 0.155 .. Phenylalanine 0.98 0.032 30 Glutamine 0 0.207 .. Methionine 2.50 0.021 120 Glycine 0 0.008 .. Tyrosine 2.99 0.067 45 Hydroxyproline 0 0.004 .. Valine 2.99 0.008 375 Lysine 0 0 .. Leucine 3.44 0.003 1150 Proline 0 0.003 .. Isoleucine 5.08 0.003 1690 * Data from J. A. DeMoss, Ph.D. thesis, Dept. of Microbiology, Western Reserve University School of Medicine, 1957. TABLE 3 COMPARISON OF RATE OF PP-ATP EXCHANGE WITH HYDROXAMATE FORMATION IN Achromobacter fisheri* RATE RATE (;MOLEs/HR/MG (LUMOLEs/HR/MG OF PROTEIN) OF PROTEIN) Ex- Hydrox- EXCHANGE Ex- Hydrox- ExCHANGE AMINO ACID change amate HYDROXAMATE AMINO ACID change amate HYDROXAMATE Alanine 0.05 0 0 Serine 0 0 0 Arginine 0.12 0 0 Threonine 0 0 0 Aspartic acid 0.19 0.43 0.44 Tryptophan 0.20 0 Asparagine 0.31 0.22 1.4 Histidine 0 0.02 0.. Glutamic acid 0.37 0.04 9.3 Phenylalanine 0 0 0* Glutamine 0.37 0.14 2.7 Methionine 2.34 0.01 2.34 Glycine 0.05 0.09 0.55 Tyrosine 0.12 0.07 1.7 Hydroxyproline 0 0 0 Valine 3.60 0 0 Lysine 0 0 0 Leucine 0 0 0 Proline 0 0 0 Isoleucine 2.34 0 0 * M. J. Cromier and G. D. Novelli, unpublished data. TABLE 4 COMPARISON OF RATE OF PP-ATP EXCHANGE WITH HYDROXAMATE FORMATION IN GUINEA-PIG LIVER* RATE RATE (pAMOLEs/HR/MG (AMOLEs/HR/MG OF PROTEIN) OF PROTEIN) Ex- Hydrox- EXCHANGE Ex- Hydrox- EXCHANGE AMINO ACID change amate HYDROXAMATE AMINO ACID change amate HYDROXAMATE Alanine 0.01 0 0 Serine 0.05 0.031 1.6 Arginine 0 0 0 Threonine 0.02 0 0 Aspartic acid 0 0.014 Tryptophan 0.06 0.037 1.6

Asparagine 0 0.20 .. Histidine 0.43 0.029 14.8

Glutamic acid 0 0.112 .. Phenylalanine 0.13 0.014 9.3 Glutamine 0 0.112 Methionine 0.55 0.015 36.6 Glycine 0.07 0.029 2.42 Tyrosine 0.19 0.025 7.6 Hydroxyproline 0.05 0.019 2.6 Valine 0.32 0.015 21.3 Lysine 0.05 0.029 1.7 Leucine 0.30 0.014 21.5 Proline 0.03 0.027 1.1 Isoleucine 0.71 0.014 50.3 Cysteine 0.06 0.019 3.2 * G. D. Novelli and A. N. Best, unpublished data.

change reaction with the rate of hydroxamic acid formation, as catalyzed by the individual amino acids, in several different extracts. These data are presented in Tables 2, 3, and 4. The points of significance are (1) the amino acids can be grouped into three classes: (a) those that catalyze only the formation of hydroxamic acid, Downloaded by guest on October 4, 2021 90 SYMPOSIUM PROC. N. A. S.

(b) those that catalyze only the exchange, and (c) those that catalyze both reactions; (2) with the amino acids that catalyze both reactions there seems to be no definite relation between the rates of the two processes. Although it is true that with the tryptophan-activating enzyme from beef pancreas9 and the tyrosine-activating enzyme from hog pancreas'0 there is a definite relation between the PP-ATP exchange and hydroxamic acid formation, the data in Tables 2, 3, and 4 suggest that, in general, the two methods may be measuring quite different reactions in the mechanism of activation of the carboxyl group. Evidence in support of this suggestion has been obtained by M. J. Cormier and Novelli (unpublished). We elected to purify the glycine-activating enzyme from Achromobacter fisheri which in a survey experiment seemed to catalyze hydroxamic acid formation without catalyzing the PP-ATP exchange. The sixteen fold purified enzyme apparently activates glycine by a process involving the elimination from ATP of one molecule of orthophosphate. Its characteristics are rather similar to those of glutamine synthetase."3 Thus it is probably unsafe to draw general conclusions about amino acid activation from observations made through the use of either method of measurement. The Mechanism of the PP-A TP Exchange Reaction.-By analogy with the pro- posal of Berg on the activation of acetate,'4 Hoagland et al.3 proposed that the PP- ATP exchange reaction occurred through the intermediate formation of an enzyme- bound aminoacyl adenylate. Support for this formulation was provided by De- Moss, Genuth, and Novelli," who synthesized leucyl adenylate and showed that it was rapidly converted to ATP in the presence of PP by an enzyme from E. coli that carried out a leucine-dependent PP-ATP exchange. These experiments suggested that such aminoacyl adenylates might indeed be intermediates in the exchange reactions and might represent the activated amino acid. However, since evidence for the formation of such compounds during the exchange reaction has not ap- peared, one cannot say with certainty that such compounds are true intermediates. Indeed, later findings have cast some doubt on the exact role of the aminoacyl adenylates in the exchange reaction. Berg'6 has reported that a methionine-activating enzyme isolated from yeast that is specific for methionine in the PP-ATP exchange reaction could nevertheless convert a variety of synthetic aminoacyl adenylates to ATP when incubated with PP. This suggested a great lack of specificity in the backward reaction. This observation was at variance with our earlier report that the leucine-activating enzyme from E. coli exhibited the same specificity in ATP synthesis from aminoacyl adenylates as it had shown in the PP-ATP exchange reaction. To explore this problem further, we tested the ability of the pan- creatic tryptophan-activating enzyme9 to form ATP from a variety of synthetic aminoacyl adenylates at various stages in the purification of the enzyme. These results are presented in Table 5. It seems clear that even the 145 fold purified enzyme (Am 65) can convert a large variety of synthetic aminoacyl adenylates to ATP. This lack of specificity extends to D compounds as well as to L,S-methyl- cysteinyl adenylate. Since ATP is not formed from carbobenzoxyleucyl adenylate or from acetyl adenylate, a free amino group in the compound seems to be necessary for activity. It does not seem reasonable that an enzyme so highly specific in one direction would be so non-specific in the backward reaction if these aminoacyl adenylates were the true intermediate in the exchange reaction. One possible ex- planation for the apparent lack of specificity for ATP synthesis with the synthetic Downloaded by guest on October 4, 2021 VOL. 44, 1958 SYMPOSIUM991

compounds might be that both the methionine-activating enzyme and the tryptophan-activating enzyme, although considerably purified, are apparently still con- taminated with a bound form of their respective amino acid, as judged by a highly significant exchange rate in the absence of added amino acid. It thus becomes possible that an anhydride exchange between the bound amino acid and the amino acid of the synthetic adenylate may occur to make the correct compound on the enzyme surface. Thus the specificity would be maintained. We are attempting to obtain experimental evidence to rationalize this point, but it is difficult to eliminate the last traces of bound amino acid from the enzyme. TABLE 5* PRODUCTION OF ATP FROM AMINO ACID ADENYLATES BY THE TRYPTOPHAN-ACTIVATING ENZYMEt AT VARIOUS STAGES OF PURIFICATION: JIMOLES OF ATP FORMED IN 15 MINUTES ADENYLAIE Sup II AC I Am 65 D-Alanine 0.083 0.125 0.111 -Alanine 0.606 0.550 0.94 D-Valine 0.570 0.525 1.04 i-Valine 0.690 0.971 1.195 D-Methionine 1.64 1.18 1.76 L-Methionine 1.18 1.76 1.23 D-Phenylalanine 1.40 1.40 1.44 L-Phenylalanine 1.67 1.20 1.68 D-Tryptophan ...... 0.75 L-Tryptophan 1.34 1.02 1.08 L,S-Methylcysteine 0.558 0.39 0.59 i-Leucine 1.002 0.945 1.078 Carbobenzoxy-ileucine 0 Acetyl 0 ... * In each case 5 emoles of compound were incubated with 60 hydroxamate units of enzyme, 50 jsmoles of KF, 10 ;&moles of ppa2 in P04 buffer. pH 6.8. t See n.9. t R. W. McRorie and G. D. Novelli, unpublished data. Support for the aminoacyl adenylates as intermediates in the activation reaction is provided by experiments with 018-labeled amino acids as detailed in another paper in this symposium (Boyer)." These experiments, however, are not decisive, since they indicate only that a physical union has occurred between the carboxyl group of the amino acid and the adenylic acid moiety of ATP, without indicating the exact chemical structure of the intermediate. Although circumstantial evidence is mounting in support of the idea that amino acid activation as reflected by the PP-ATP exchange reaction is involved in protein biosynthesis, the present observations suggest that more data are necessary before a definitive statement can be made. * Operated by Union Carbide Nuclear Company for the U.S. Atomic Energy Commissiori. I M. B. Hoagland, Biochim. et Biophys. Acta, 16, 288-289, 1955. 2 J. A. DeMoss and G. D. Novelli, Biochim. et Biophys. Acta, 18, 592-593, 1955. 3 M. B. Hoagland, E. B. Keller, and P. C. Zamecnik, J. Biol. Chem., 218, 345-358, 1956. 4 G. D. Novelli and J. A. DeMoss, J. Cellular Comp. Physiol., 50 (Suppl. 1), 173-107, 1957. 5 N. Sharon and F. Lipmann, Arch. Biochem. and Biophys., 69, 219-227, 1957. 6 F. Lipmann, these PROCEEDINGS, this symposium. I P. Zamecnik, these PROCEEDINGS, this symposium. 8 E. J. Cohn and J. T. Edsall, Proteins, Amino Acids and Peptides (New York: Reinhold Publishing Co., 1943). 9 E. W. Davie, V. V. Koningsberger, and F. Lipmann, Arch. Biochem. and Biophys., 65, 21-38, 1956. Downloaded by guest on October 4, 2021 92 SYMPOSIUM PROC. N. A. S.

10 R. S. Schweet, R. W. Holley, and E. H. Allen, Arch. Biochem. and Biophys., 71, 311-325, 1957. 11 V. V. Koningsberger, A. M. Van De Ven, and J. T. G. Overbeek, Koninkl. Ned. Akad. Weten- schap., Proc., Ser. B, 60, 141-143, 1957. 12 P. Berg, J. Biol. Chem., 222, 1025-1034, 1956. 13 L. Levintow, A. Meister, G. H. Hogeboom, and E. L. Kuff, J. Am. Chem. Soc., 77, 5304-5308, 1955. 14 P. Berg, J. Biol. Chem., 222, 991-1014, 1956. 15 J. A. DeMoss, S. M. Genuth, and G. D. Novelli, these PROCEEDINGS, 42, 325-332, 1956. 16 P. Berg, Federation Proc., 16, 152, 1957. 17 P. Boyer, these PROCEEDINGS, this symposium.

TRACING OF THE IN VIVO PATH FROM AMINO ACID TO PROTEIN* P. D. BOYER AND M. P. STULBERG DEPARTMENT OF PHYSIOLOGICAL CHEMISTRY, UNIVERSITY OF MINNESOTA, MINNEAPOLIS 14, MINNESOTA The problem of protein synthesis has many facets. The previous speakers on this symposium have outlined and discussed the excellent researches with cellular fractions and partially purified enzyme systems which demonstrate activations and incorporations of amino acids dependent upon energy derived from cleavage of ATP. Information from these studies suggests that similar mechanisms operate in vivo and thus that, for every peptide bond formed, at least one ATP molecule is split. Over the past several years, studies have been made in our laboratory on the mechanisms of ATP utilization and formation in various reactions catalyzed by cell fractions and purified enzymes. An important part of these researches has dealt with the fate of the oxygen atoms in substrate molecules participating in the coupled reactions as studied by use of the heavy isotope of oxygen, 018. These and related studies by other investigators have yielded fundamental information about the course of the reactions and have suggested, as will be outlined presently, that it might be possible to put to test the hypothesis that protein synthesis in the intact cell involves cleavage of an ATP molecule for each peptide bond formed, through participation of an intermediate formed by combination of part of the ATP molecule with the carboxyl group of the amino acid. The purpose of this contri- bution is to present results of studies on the fate of the carboxyl oxygens of amino acids, which allow tracing the path of amino acids to protein in an intact cell, and to deduce some aspects of the nature of the primary reactions and other reactions associated with the protein synthesis. When an ATP molecule is cleaved to ADP and iP (inorganic orthophosphate), or to ADP and PP (inorganic pyrophosphate), an oxygen atom from some source must be incorporated into the products. In some metabolic processes, such as muscular contraction, hydrolysis of ATP appears to be involved, and the oxygen would be furnished by water. In syntheses coupled to ATP degradation, the pos- sibility exists that the required oxygen would be furnished by one of the substrates participating in the over-all reactions. That such is indeed the case is shown by the researches summarized in Table 1. This table gives the results of 018 studies Downloaded by guest on October 4, 2021