Tue Journau or Biotogican CHEMISTRY
Vol, 236, No. 6, June 1961
Printed in U.S.A

The Enzymic Synthesis of Amino Acyl Derivatives
of Ribonucleic Acid

I. THE MECIIANISM OF LEUCYL-, VALYL-, ISOLEUCYL-, AND METHIONYL RIBONUCLEIC
ACID FORMATION*

Pact Bere,t Frep H. Beramann,t E. J. Orencann,§ anp M. Dieckmannt

From the Department of Microbiology, Washington University School of Medicine, St. Louts 10, Missouri

(Received for publication, November 8, 1960)

The enzymic formation of enzyme-bound amino acyl adenyl-
ates from adenosine triphosphate and amino acid (Equation 1)
has been recognized for several years (1-8) and enzymes specific
for certain of the amino acids have been isolated in a number of
laboratories (2, 9, 10). These same enzymes are now known
(11-15) to catalyze a second reaction involving the transfer of
the amino acyl moiety from the adenosine phosphate moiety to a
specific type of ribonucleic acid (Equation 2). The over-all
reaction catalyzed by such amino acyl ribonucleic acid synthet-
ases' is summarized in Equation 3,

 

- Mgt*
AMP-PP + RCHNH.,COOH + enzyme ——=—>
1 ”
enzyme—AMPCCHNH2R + PP;
1
Enzyme—AMPCCHNELR + RNA—OH =
0 (2)
|
RNA-OCCHNH.R + AMP
Mgt
AMP-PP + RCHNH,COOH + RNA--OH —=—
(3)

0
|
RNA—OCCHNER + AMP + PP

 

* This investigation was supported by grant funds from the
National Institutes of Health of the United States Public Health
Service.

7 Present address, Department of Biochemistry, Stanford Uni-
versity School of Medicine, Palo Alto, California.

t Postdoctoral Research Fellow of the National Institutes of
Health, United States Public Health Service; present address,
Department of Biochemistry, Brandeis University, Waltham,
Massachusetts.

§ Predoctoral Research Fellow of the National Science Founda-
tion; present address, Medical Research Council Unit, Cavendish
Laboratory, Cambridge University, Cambridge, England.

!Tnzymes which catalyze an amino acid-dependent ATP-PP
exchange and ATP-dependent amino acid hydroxamate formation
have been referred to as amino acid-activating enzymes (1). In-
asmuch as these activities are partial manifestations of the over-all
reaction leading to amino acyl RNA formation (12, 16, 17) we
propose to designate this class of enzymes as amino acyl RNA
synthetases and the enzyme specific for a single amino acid, e.g.
leucine, as leucyl RNA synthetase. This nomenclature, we feel,
is consistent with the practice of including some indication of the
nature of the product formed in the reaction. Moreover, it
minimizes any ambiguity arising from situations in which amino
acid activation occurs by reactions not involving amino acyl RNA

By the above reaction, the amino acids are bound to the ac-
ceptor ribonucleic acid through an ester linkage to the 2’- or
3’-hydroxyl group of the terminal nucleotidy! ribose moiety (23-
25), and where this has been examined, each amino acid is linked
to a terminal adenylic acid (16, 23-25).

The results of our investigations on the mechanism of amino
acyl ribonucleic acid formation are reported in the present com-
munication. The purification and characterization of the specific
amino acyl ribonucleic acid synthetases and the amino acid-
acceptor ribonucleic acid from /scherichia coli are presented in
the following papers (26, 27). The fourth communication (28)
describes the enzymic removal and resynthesis of the 3’-hydroxy-
ended trinucleotide portion of the acceptor ribonucleic acid.

EXPERIMENTAL PROCEDURE

Materials

Enzymes-~-In some of our earlier studies and in several experi-
ments reported here, extracts of #. colt and a mixture of uni-
formly C-labeled amino acids were used as a means of gener-
ating highly labeled amino acyl adenylates. Extracts were
prepared from cells grown as described in Paper II (26) by treat-
ment of a washed cell suspension (4 ml of 0.05 m glycylglycine
buffer, pH 7.0, per g wet weight of cells) in a cooled Raytheon
10 ke sonic oscillator for 15 minutes or by disruption in a War-
ing Blendor with glass beads (26). Both types of extract were
dialyzed for about 24 hours against 30 to 40 volumes of 0.01 m
Tris buffer, pH 8.0.

The leucyl-, valyl-, isoleucyl-, and methionyl RNA synthetases
from FE. colt were prepared as described in Paper II of this series
(20); the isolation of the methionyl RNA synthetase from yeast
has been reported previously (2, 29).

Crystalline inorganic pyrophosphatase (30) was kindly sup-
plied by Drs. G. Perlmann and M. Kunitz.

Amino Acid-Acceplor RNA Preparations—The acceptor RNA
was isolated as described by Ofengand eé al. (27), and in almost
all cases, the material eluted from Mcteola (Brown Company) was
used. The concentration of the acceptor RNA is expressed in
terms of its nucleotide content and determined by its optical
density at 260 my in 0.01 n KOH with a value of 10.0 as equal
to 1 pmole of RNA nucleotide.

 

formation, e.g. S-adenosyl methionine (18), glutamine (19), gly-
cineamide ribonucleotide (20, 21) formation, and very likely the
formation of peptides (22).

1726
June 1961

C'labeled Amino Acids—The uniformly C'*-labeled amino
acid mixture was obtained from the protein of Chromatium
grown in the presence of NaHC™O; as carbon source (31). The
protein was hydrolyzed in 6 N HCl at 110° for 18 hours. The
specific activity of the amino acids was 2.5 to 3.0 x 10° c.p.m.
per wg atom of carbon. pxi-Leucine-1-C™, pi-valine-1-C™, and
1-methionine-CH3-C™ were purchased from Isotope Specialties,
Inc., and uniformly labeled L-isoleucine-C* was obtained from
Volk Radiochemical Company. The specific activities of the
amino acids ranged from 3 to 17 X 106 c¢.p.m. per wmole counted
in a windowless gas flow counter.

Miscellaneous—PP;* was made as previously described (29).
Nucleoside mono-, di-, and triphosphates were obtained from
the Sigma Chemical Company, and unlabeled amino acids were
purchased from the California Foundation for Biochemical Re-
search or from Nutritional Biochemicals. As pointed out else-
where (26), ib was necessary in certain cases to use synthetic
preparations of the amino acids to avoid trace contaminations
by other amino acids.

Methods

Measurement of Amino Acyl RNA Formation—Depending
upon the experiment, one of two assays for amino acyl RNA
formation was carried out. The first determined the yield of
amino acyl RNA formed when the enzyme, ATP, and amino
acids were present in excess and the amount of acceptor RNA
was limiting. The standard conditions for this measurement
were as follows. The incubation mixture contained in a total
volume of 0.5 ml, 50 umoles of sodium cacodylate buffer, pH 7.0;
0.5 umole of ATP; 1.0 wmole of MgCl, (for leucyl- and valyl
RNA formation) or 5.0 wmoles of MgC (for isoleueyl- and
methiony! RNA formation); either 0.8 wmole of pi-leucine-1-C%,
0.4 umole of pt-valine-1-C™, 0.03 umole of uniformly labeled
L-isoleucine-C™ or 0.3 umole of L-methionine-CH;-C™; 0.2 to 1.0
umole of acceptor RNA nucleotide; 100 ug of crystalline beef
serum albumin; 2 wmoles of reduced glutathione; 5 umoles of
potassium chloride (for methionyl RNA formation); and either
0.9, 0.5, 7, or 3 wg of protein of the leucyl-, valyl-, isoleucyl-, or
methiony! RNA synthetase preparations, respectively. The
mixture was incubated at 30° for 20 minutes (a time which was
sufficient for the reaction to come to completion) and the reac-
tion was stopped by the addition of 0.5 to 1.5 mg of carrier
yeast RNA and 3 mi of a cold solution containing 0.5 m NaCl
and 67% ethanol. After 5 minutes at 0°, the precipitate was
centrifuged and washed three times by resuspension in the
ethanol-salt mixture. The precipitate was dissolved in 1 ml of
1.5 n NH,OH, and a suitable aliquot was dried in small dishes
and counted in a windowless gas flow counter. The results are
expressed as millimicromoles of amino acid bound per umole of
acceptor RNA nucleotide. Data to be presented below (Fig. 5)
show that, under these conditions, the amount of each of the
amino acids bound is proportional to the amount of acceptor
RNA added.

In contrast to the first assay, which determined the yield of
product, the second assay measured the rate of amino acyl RNA
formation and was carried out under the conditions described
above, except with less enzyme and more acceptor RNA (1.0 to
2.0 wmoles of RNA nucleotide). The reaction rate was propor
tional to enzyme concentration over the range shown in Fig. 1.

Measurement of Amino Acyl Adenylate Formation—The capac-
ity of each of the enzymes to form amino acyl adenylates was

P. Berg, F. H. Bergmann, E. J. Ofengand, and M. Dieckmann

 

 

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Fic. 1. Linear relationship of the rate of amino acyl RNA for-
mation with enzyme concentration. See text for conditions.

measured by the amino acid-dependent exchange of ATP and
PP; (26). Inasmuch as the rate of amino acyl adenylate for-
mation was the rate-determining step in the over-all exchange
reaction (2, 29), the amino acid-dependent incorporation of
PP;” into ATP actually measured the rate of amino acyl adenyl-
ate formation.

For comparisons of amino acy! adenylate and amino acyl RNA
formation under the same conditions, the following assay was
used. In a volume of 1.0 ml were 100 wmoles of sodium caco-
dylate buffer, pH 7.0, 5 wmoles of MgCh, 2 pmoles of ATP, 2
uwmoles of PP; (specific activity, 0.5 to 1.0 X 105 ep.m. per
umole), 2 umoles of the 1-form of leucine, valine, isoleucine, or
methionine, 200 ug of serum albumin, 4 wmoles of recluced gluta-
thione (where indicated above), 10 umoles of KCl (where indi-
cated above), and enough enzyme to give an incorporation of
0.01 to 0.3 umole of PP? into ATP. The mixture was incubated
at 30° for 15 minutes and the ATP was isolated and counted as
previously described (2). All values were corrected for any
ATP formed in the absence of amino acid. This blank was
always less than 5% of that observed with amino acid.

RESULTS

Required Components for Enzymatic Synthesis of Amino Acyl
RNA Compounds—Formation of the amino acyl RNA derivatives
was observed in the presence of ATP, Mg**, a specific RNA
1728

Tasie I

Requirements for amino acyl RNA formation by
amino acyl RNA synthetases from E, coli
The incubation mixtures and conditions used for measuring
the rate of formation of each amino acyl RNA derivative are de-
scribed under ‘‘Methods.’’ The column headings refer to the iso-
lated enzymes which are relatively specific for the amino acids
listed (26).

Enzymic Synthesis of Amino Acyl RNA Derwatives. I

 

 

 

Components Leucine | Valine | Isoleucine | Methionine
amoles/mg/hour
Complete.............. 3.1 21.0 | 3.3 3.3
Minus ATP............ <0.03 | <0.2 <0.02 0.1
Minus RNA........... <0.03 <0.2 <0.02 <0.1
Minus Mgtt........... 0.33 0.6 0.15 <0.4
Minus enzyme......... <0.03 <0.2 <0.02 <O.1

 

 

 

 

 

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2 4 6 8 0 12 4 16 18
Time in minutes

Fie. 2. Reversibility of amino acyl RNA synthesis. The Ct
labeled mixed amino acyl RNA was prepared by incubating 2000
umoles of cacodylate buffer, pH 7.0, 90 umoles of MgCle, 8 umoles
of ATP, 1.17 X 10° c.p.m. of the C'4-amino acid mixture, 93 umoles
of acceptor RNA, and 8.0 mg of protein of a sonic extract of Z.
colt in a volume of 20 ml for 60 minutes at 30°. The product was
isolated by the addition of NaCl to a concentration of 1.5 m fol-
lowed by 2 volumes of cold ethanol. After chilling the mixture,
the precipitated product was removed by centrifugation and ex-
tracted with 0.01 M cacodylate buffer, pH 7.0. Denatured protein
was removed by centrifugation, and the process of precipitation,
buffer extraction, and removal of denatured protein was repeated
twice more. ‘The final product was dissolved in 0.02 m succinate
buffer, pH 6.

The amino acyl RNA was incubated with 20 umoles of cacodylate
buffer, pH 7.0, 15 umoles of MgCl: and, where indicated, approxi-
mately 2 umoles of C!?-amino acid mixture prepared from Chroma-
tuum, 2 umoles of AMP, 2 wmoles of PP;, 4 nmoles of P;, and 0.2
mg of protein of a sonic extract of FH. coli in a volume of 0.5 ml,
for the indicated time at 30°. The amount of amino acid remaining
bound to the RNA was determined by measuring the amount of
C4-amino acid still precipitable by 0.6 m perchloric acid. The
abbreviation used is: Enz, enzyme.

Vol. 236, No. 6

Tape IT
Formation of ATP from valyl RNA, AMP, and PP

The complete system contained, per ml, 100 wmoles of sodium
cacodylate buffer, pH 7.0, 2 zmoles of MgClz, 50 umoles of potas-
sium fluoride, 200 »g of serum albumin, 2.388 mumoles of valinc-
C# as valyl RNA, 106 ug of valyl RNA synthetase protein, 0.10
umole of AMP, and 0.06 pmole of PP; (8.4 X 108 ¢.p.m. per
umole). Valyl RNA was hydrolyzed to free acceptor RNA and
valine by heating at 55° for 15 minutes at pH 9. The incubation
was at 30° for 15 minutes and the reaction was stopped by boiling
for 2 minutes. An aliquot of the reaction mixture was removed
and the amount of valyl RNA remaining was determined by the
amount of radioactivity still precipitable after the addition of the
NaCl-ethanol mixture described under ‘‘Methods.”’ After the
addition of unlabeled ADP, ATP, and PP; to the remainder of
the reaction mixture, the nucleotides were adsorbed on charcoal.
After the charcoal was washed several times with 0.01 m PP,
the nucleotides were eluted with 50% ethanol containing 0.3 m
NH,OH and chromatographed on a Dowex 1-Cl~ column (32).
In control experiments in which PP; was omitted, there was no
disappearance (<5%) of valyl RNA or ATP when added in the
amounts obtained in the experiment. Over 90% of the radioac-
tivity was eluted with the carrier ATP, whereas in the experi-
ment with hydrolyzed valyl RNA, less than 5% of the P® appeared
with the ATP. In the former case, the specific activity of the
ATP was essentially constant over the entire peak. The isolated
material was further identified as ATP by the following two ex-
periments. After reaction of the ATP® with glucose and hexo-
kinase, 45% of the P*? was isolated in the glucose 6-phosphate
and 55% in the ADP. With an excess of valyl RNA synthetase,
L-valine, and unlabeled PP;, under conditions of the ATP-PP ;#
exchange reaction (26), 95% of the P® in the ATP was found in
the PP; fraction.

 

 

 

Valyl RNA ATP
Conditions 7
Initial Final | A Final A
mymoles mymoles

Complete system....| 1.19 0.06 —1.13 1.11 +1.06
Complete system

with hydrolyzed

valyl RNA........ 0.04 0.05

 

fraction isolated from HE. coli, a C-labeled amino acid, and the
purified enzyme fraction capable of converting that amino acid
to the corresponding amino acyl adenylate (Table I). In each
case, omission of any one of the cited components resulted in a
marked decrease in the rate of amino acyl RNA synthesis.
Substitution of the ATP by UTP, GTP, CTP, dATP, or ADP
lead to a decrease in the rate to less than 1%. Ribosomal RNA
(27) from F. colt, or the equivalent fractions from animal and
other bacterial sources, and synthetic polynucleotides prepared
with polynucleotide phosphorylase (81), failed to function as
amino acid acceptors under these conditions (32).

Reversibility of Amino Acyl RNA Formation—Equation 3 pre-
dicts that amino acyl RNA formation is reversible. Substantia-
tion of this prediction was given by the following experiments.
When amino acid-acceptor RNA, to which a mixture of Cl.
labeled amino acids had been linked, was incubated with AMP,
PP; and a dialyzed extract of F. colt, there was a rapid removal
of the labeled amino acids from the RNA (Fig. 2). This occurred
whether or not a pool of unlabeled amino acids was added. If
either the AMP, PPi, or the extract was omitted, or if UMP or
CMP replaced AMP, or if P; was substituted for PP:, the rate
June 1961 P. Berg, F. H. Bergmann, E. J. Ofengand, and M. Dieckmann 1729

Tasir ITI
Determination of equilibrium constant of L-valyl RNA formation
For each experiment, 350 umoles of sodium eacodylate buffer, pH 7.0, 7 umoles of MgCls, 0.7 mg of serum albumin, 175 umoles of
KF, and the reactants shown in the table were incubated in a volume of 3.5 ml. The valyl RNA was labeled with L-valine-1-C' (6 X
10° ¢.p.m. per umole). Samples were removed at 2,3, 4,5, 10,15, and 20 minutes and the valy] RNA formed or remaining was deter-
mined as described in the standard assay procedure. In each case, the reaction was followed until no further change in the amount
of valyl RNA could be detected. Completion of the reaction occurred by 5 minutes, and no change was measurable up to 20 minutes.
The concentration of RNA is expressed as millimicromoles of valine-specific acceptor sites. The concentration of the RNA was
calculated by the difference between the amount of valine-specific acceptor RNA added and the amount of valyl RNA formed or from

the amount of valyl RNA which disappeared.

Inasmuch as the concentrations of each of the other components was large compared
to the amount of reaction which had occurred, the initial concentrations of each were used in the calculation.

In separate experi-

ments, it was shown that under these conditions there was no detectable destruction of the valine acceptor RNA chains nor was there
any disappearance of ATP, AMP, or PP; (<4%) when added separately.

 

Initial concentrations

Experiment No.

Final

concentrations K*

 

AMP | PP} | ATP | Valine

 

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0.

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ooo
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oe - (AMPUPP)Valyl RNA)
* = “(CATP) (Valine) (RNA)

 

a

Valyl RNA, moles /mi. x 10°
|

 

 

| J i
iO 1S
Time in minutes

Fig. 3. The equilibrium position for the valine incorporation
reaction. The incubation conditions are described in Table III.
O-——6O, experiment 1; O——©, experiment 2; O——O, experi-
ment 3; @——-@, experiment 4.

Lp} ttt
20

 

of amino acid removal from the RNA occurred at less than 1%
the rate. The failure to remove amino acids from the RNA in
the presence of only AMP and the enzyme was also consistent
with the formation of enzyme-bound amino acyl adenylates by
the reverse as well as the forward reaction (7, 8).

ValyiRNA| Valyi RNA

RNA synthetase

Valyl] RNA |

pmoles/ml X 108
2.4

units/ml
2.3
1.2
0.3
0.3.

1.36 * 1078 0
1.36 X 1073 0

0 1.36 « 107%
1.36 X 1073 0

0.36
5.4 0.32
4.8 0.28
5.1 0.30

0.32

 

That the previous observations do represent reversal of amino
acyl RNA synthesis was established by the finding that incuba-
tion of C-valyl RNA with AMP, PP;*, and the specific valyl
RNA synthetase resulted in essentially complete removal of the
valine from the RNA and stoichiometric formation of ATP#
(Table II). No ATP formation is observed when an equivalent
amount of RNA and valine is substituted for the valyl] RNA.

Determination of the equilibrium constant for valyl RNA
formation was made by measuring the steady state concentra-,
tion of valyl RNA in the presence of the other components of
the system (Table III and Fig. 3). The average K.q value of
0.32? showed that there was little change in free energy resulting
from the formation of valyl RNA at the expense of the cleavage:
of ATP.

Existence of Specific Acceptor RNA for Each Amino Actd—An
examination of the kinetics of amino acyl RNA formation showed
that the reaction proceeded linearly with time and then reached
a limit (Fig. 4). This limit was not appreciably increased (less
than 5%) by the addition of up to 5 times more enzyme, ATP,
or amino acid, whether added initially or when the reaction had
stopped. The addition of 2.6 wy of crystalline inorganic pyro-
phosphatase did not affect the extent of amino acyl RNA forma-
tion. However, the addition of acceptor RNA, either at the
beginning of the reaction or at the time the reaction ceased, lead
to an increased yield of amino acyl RNA. If in each case the
reaction was allowed to proceed to completion in the presence of
varying amounts of acceptor RNA, the amount of amino acyl
RNA formed was a linear function of the amount of acceptor
RNA added (Fig. 5). It should be noted, however, that the
yield of each amino acyl RNA was different. Thus, although

2 It should be pointed out that the calculation of the Ke, does
not take into account the concentrations of possible complexes of
the phosphorylated derivatives (e.g. ATP, RNA, etc.) with Mg**.
 

9
a
|
L

oO
uy
I

mumoles of amino acyl RNA forrned
a K
T
|

2
ty
I

ot -

[

 

 

! |
20 30
Time in minutes

0.0

 

or

0

Fre. 4. Kinetics of amino acyl RNA formation. The condi-
tions used were those described for the usual assay of amino acyl
RNA formation except that 1.2, 0.1, 0.4, or 0.5 ug of leucyl-(Z),
valyl-(V), isoleucyl-(£), or methionyl-(47) RNA synthetase pro-
tein, respectively, and 1.0 pmole of acceptor RNA nucleotide were
used.

 

OV

O° °
& K
T qT
l L

moles of amino acyl RNA formed
S
|

m
of
j

   

ZA .
fi

i L ! i ! i t {
0.0 0.2 0.4 -0.6 0.6 1.0
Almoles of accepior-RNA nucleotide added

 

 

|

 

Fie. 5. Formation of amino acyl RNA as a function of the
amount of acceptor RNA. The conditions used were those de-
scribed under ‘‘Methods.”’ Abbreviations are as in Fig. 4.

Enzymic Synthesis of Amino Acyl RNA Derivatives. I

Vol. 236, No. 6

TaBLe IV
Separate sites for linking amino acids to acceptor RNA

Experiment 1. The reaction mixture (0.5 ml) contained 20
umoles of sodium cacodylate buffer, pH 7.0, 1 wmole of MgCla, 0.2
umole of ATP, either 0.25 wmole of pi-leucine-1-C'4 (5.1 XK 108
c.p.m. per zmole), 0.25 pmole of pL-valine-1-C* (6.1 X 10° ¢.p.m.
per pmole) or 0.16 pmole of L-methionine-CH;-C% (3.0 X 108
c.p.m. per umole), 0.9 pmole of acceptor RNA, and 100 ug of a
sonic extract of #. coli. The incubation was for 20 minutes at
30°. In the experiment with a mixture of the three amino acids,
all were present initially.

Experiment 2. The incubation mixtures and conditions were as
described under ‘‘Methods.’’? When the incorporation of two
amino acids was examined, the second amino acid and the appro-
priate enzyme were added after 20 minutes, and the incubation
was continued for an additional 20 minutes.

 

 

Experiment Amino acid added Incorporation
total c.p.m.
1 Leucine 3759
Valine 1646
Methionine 468
Mixture of above 5832
Calculated sum 5873
2 Valine 947
Leucine 1038
Valine, then leucine 2006
Calculated sum 1985
Valine 947
Methionine 448
Valine, then methionine 1434
Calculated sum 1395

 

 

 

the RNA acted stoichiometrically with each amino acid, for a
given amount of acceptor RNA the amount of amino acyl RNA
formed varied with the amino acid.

Two possible interpretations of this result are that (a) there
was a single binding site which reacted with each amino acid to
a different extent, or (6) there existed different and specific sites
for the individual amino acids. These alternatives were dis-
tinguished by the following experiments (Table IV). When
leucine, valine, and methionine were present together, the total
amount of amino acid linked to the acceptor RNA was equal to
the sum of the amounts obtained when each amino acid was
present by itself (Experiment 1). Moreover, saturation of the
acceptor RNA with one amino acid (e.g. L-valine) did not affect
the amount of any other amino acid (e.g. L-leucine or L-methio-
nine) which could subsequently be linked to the acceptor RNA
(Experiment 2). These data ruled out the common binding site
hypothesis but were consistent with the existence of a limited
and fixed number of binding sites, each specific for a particular
amino acid.

Further support for this view has come from studies on the
destruction of amino acid acceptor sites by periodate (28). This
work showed that periodate oxidation of acceptor RNA destroys
the ability to accept all amino acids. However, similar treat-
ment of leueyl-, valyl-, or methionyl RNA followed by removal
of the amino acid yielded preparations of RNA which could
accept only that amino acid which was linked to the RNA during
the exposure to periodate. Simce, according to currently ac-
cepted ideas of RNA structure, polynucleotide chains are un-
June 1961

branched and therefore the only cis-hydroxyl configuration re-
sides on the terminal nucleotide with a free 3’-hydroxyl group,
it may be inferred that each amino acid is linked exclusively to
either the 2’- or 3’-hydroxy] group of the terminal nucleotidy]
ribose unit of individual RNA molecules. For the leucine- and
valine-specific polynucleotide chains this terminal nucleotide is
adenylic acid (28), although it is now clear that, in the acceptor
RNA of #. coli, adenylic acid is the sole terminal nucleotide
containing a free 8’-hydroxyl group on the ribose moiety (27).

Heterogeneity of Acceptor RNA Chains Reacting with Single
Amino Acid—The conclusion stated above predicts that acceptor
RNA represents a heterogeneous population of polynucleotide
chains, each chain being specific for a particular amino acid. It
is essential before considering any analysis of the chemical basis
of the amino acid specificity of acceptor RNA to know whether
there exists a second order of heterogeneity, namely, whether all
the chains reacting with a particular amino acid are identical.
The following experiments suggested that they are not.

Acceptor RNA from £. coli bound methionine to a different
extent depending upon whether the methionyl RNA synthetase
from LE. colt or yeast was used (Fig. 6). Although the amount
of methionine fixed was a direct function of the acceptor RNA
added, the slopes of the two curves differed by a factor of
about 2.5; that is, 2.5 times more methionine was bound per unit
of RNA when the synthetase from Z. coli was used as compared
with the one from yeast. Although the addition of more yeast
methionyl RNA synthetase, ATP, or methionine did not in-
crease the yield of methionyl RNA, it was clear that there still
were sites available to accept methionine. This is shown by the
experiment (Table V) in which the 2. coli synthetase was added
when the reaction with the yeast enzyme had come to completion.
The reciprocal experiment, in which the acceptor RNA was re-
acted to a limit with methionine with the #. colt enzyme and then
exposed to the yeast enzyme, showed no additional formation of
methionyl RNA. It may be inferred from this result that of
the polynucleotide chains specific for methionine, 40% can func-
tion with either enzyme, whereas 60% of the chains are available
only to the £. cole synthetase.

Support for this interpretation was obtained by the periodate
oxidation technique for selectively inactivating those polynucleo-
tide chains not linked to amino acids (28). Samples of methionyl
RNA prepared with the #. colt or yeast synthetases were treated
with periodate, reisolated, and then the amino acids were re-
moved with alkali. The regencrated acceptor RNA preparations
were retested for their capacity to accept methionine with each
of the enzymes (Table VI). When the methionyl RNA was
prepared with 2. colt enzyme, all sites specific for methionine
survived the periodate oxidation when tested with either enzyme.
On the other hand, when methionyl RNA was prepared with the
yeast cnzyme, 60% of the chains which accept methionine were
inactivated as judged by the test with the enzyme from F. colt
but all were conserved when assayed with the yeast enzyme.
These data show that within the population of acceptor RNA
molecules there were at least two distinguishable classes of poly-
nucleotide chains which could accept methionine.

Nature of Enzymes Catalyzing Amino Acyl RNA Formation—
The enzyme preparations used in the present studies were puri-
fied on the basis of their activity for amino acyl adenylate forma-
tion (26). Although these same preparations catalyzed the
formation of the amino acyl RNA derivatives, it was not clear
which of the following hypotheses was operative.

1. The formation of the specific enzyme-amino acy! adenylate

P. Berg, F. H. Bergmann, EF. J. Ofengand, and M. Dieckmann

 

 

 

 

1731
T T T T T T T T

3

Fook 4
2 E.col/

<

=

oO
ZS

€

o

g

°°

£01 EK e
% yeast

a

2

e ° 6

g

0.0 | L | | { ! i t
0.0 0.2 O4 0.6 0.8 1.0

Admoles of acceptor-RNA nucleotide added

Fic. 6. Formation of methionyl RNA by methionyl RNA syn-
thetases from yeast and #. colz. The conditions used are those
described under ‘‘Methods.”’

TaBLE V

Formation of methionyl RNA by methionyl RNA synthetases from
E. colt and yeast

The reaction mixtures contained, in a volume of 0.5 ml, 50
umoles of sodium cacodylate buffer, pH 7.0, 1 umole of MgCla, 0.5
umole of ATP, 0.06 umole of L-methionine-CH;-C™ (7.5 x 108
¢.p.m. per umole), 5 zmoles of potassium chloride, 100 ng of serum
albumin, 0.81 wmole of acceptor RNA, and either 30 ug of protein
of methionyl RNA synthetase from &#. coli or 125 wg of protein
of the similar enzyme from yeast added as indicated in the table.
The incubation was at 30°.

 

 

 

 

Total time} Methionyl
Enzyme additions of RNA
incubation formation
min | Munole/umal
FY. coli enzyme at time zero............... 20 0.28
40 0.27
£. coli enayme at time zero and again at 20
MINUEES. 6 eee eee 40 0.28
H. colt enzyme at time zero; yeast enzyme .
at 20 minutes..........0..... 00.002 eee 40 0.28
Yeast enzyme at time zero................ 20 0.12
40 0.14
Yeast enzyme at time zero and again at 20
minutes. ......0. 0. ee eee 40 0.13
Yeast enzyme at time zero; #. colt enzyme
at 20 minutes............0 0... ee eee eee 40 0.28
Yeast and #. colt enzyme at time zero... | 20 0.27

1

 

complex may be followed by a nonenzymic transfer of the amino
acid to the RNA.

2. There may be a single amino acid-specific enzyme which
catalyzes the formation of an amino acyl adenylate and the
transfer of that amino acid from the adenylate moiety to the
acceptor RNA.

3. There may be required in addition to the enzyme forming
each amino acyl adenylate either (a) separate specific amino acy!
transferases for linking each amino acyl residue to the appro-
priate acceptor RNA, or (b) a single amino acyl transferase which
1732

TaBLe VI
Evidence for heterogeneity among acceptor RNA
chains for L-methionine

Methionyl RNA was prepared under the usual conditions with
either the methionyl RNA synthetase from #. colt or from yeast
and was isolated as already described. Bothsamples were treated
with sodium metaperiodate in 0.1 m sodium suecinate buffer at
pH 5.6 in the ratio of 1 umole of periodate per 14 ymoles of ac-
ceptor RNA nucleotide. The RNA samples were reisolated and
the bound methionine removed by 0.1 m glycine buffer, pH 10.2,
at 30° for 60 minutes. The two RNA preparations were again
recovered and then tested as acceptors of methionine with each
of the methionyl RNA synthetase preparations as described in
Table V.

 

Methionyl RNA formation

 

Source of enzyme used
to measure methionyl
RNA formation

Periodate-treated
' methionyl RNA prepared with
Original RNA :

Yeast enzyme

 

| &. coli enzyme

 

myumoles/pmole RNA nucleotide
0.23
0.10

0.10
0.10

 

 

transfers any amino acyl group but only to the appropriate ac-
ceptor RNA chain.

Although nonenzymic acylation of RNA by amino acy] adenyl-
ates has been observed, the amino acids appeared to be bound
to any RNA and in a variety of linkages (34). Such a mecha-
nism would therefore not account for the fact that only a particu-
lar fraction of RNA functions as an amino acid acceptor (11, 27).
It also seems unlikely that a nonenzymic mechanism would
manifest the high degree of specificity inherent in linking each
amino acid exclusively to the terminal nucleotide of a particular
RNA chain. Furthermore, the different yields of methionyl
RNA produced in the presence of two different methionyl RNA
synthetases are inconsistent with a nonenzymic transfer reaction.

Since hypotheses 2 and 3 predict that the synthesis of a given
amino acyl RNA derivative is preceded by the formation of the
corresponding amino acyl adenylate, it is implicit in cither al-
ternative that the formation of each amino acyl RNA compound
must be at least as specific with respect to the amino acid as is
the synthesis of the amino acyl adenylate. Table VII shows
that with four cnzymes from £. coli and one from yeast only
that amino acid which is converted to the adenylate is linked to
the acceptor RNA. The only deviation from an exact correla-
tion of the two specificities is the case of the isoleucyl RNA
synthetasc. Although this enzyme can form both isoleucyl- and
valyl adenylates (26), it synthesizes only isoleucyl RNA. The
reasons why the valyl moiety is not transferred to the acceptor
RNA (which can accept valine from the valyl RNA synthetase
preparation) remain to be determined.

Hypothesis 2, in contrast to 3, predicts that the ratio of the
activity for amino acyl adenylate formation and amino acy] RNA
synthesis must be constant throughout the purification of the
enzymes. Any alteration in this ratio during purification would
suggest the existence of separable activities. Table VIII shows
that the ratio of activities for methiony] adenylate and methionyl
RNA formation was constant during the course of an approxi-
mately 100-fold purification of the methionyl RNA synthetase
from yeast. Similar findings have been made with the leucyl-
and methionyl RNA synthetases of #. coli. The ratio of leucy!

Einzymic Synthesis of Amino Acyl RNA Derivatives. I

Vol. 236, No. 6

TaBLe VII
Specificity of enzyme preparations for amino acyl adenylate and
amino acyl RNA formation
The rate of amino acyl adenylate formation was measured as
described elsewhere (26), and the rate of amino acyl RNA synthe-
sis was determined as described under. ‘‘Methods.”’

 

 

 

 

 

 

Amino acyl | Amino acyl
Enzyme Amino acid tested adenylate RNA
formation formation
umoles/mg/hour
Leucine Leucine 358 3.2
Valine 13.2 0.18
Methionine 8.0 <0.01
Isoleucine <3.0 <0.01
Valine Valine 560 25
Leucine <0.5 <0.01
Isoleucine <4.0 <0.01
Methionine 2.0 <0.01
Tsoleucine Isoleucine 768 3.3
Leucine 31 <0.07
Valine 416 <0.03
Methionine 41 0.07
Methionine (Z#. colt) Methionine 356 3.5
Leucine 4.0 <0.01
Valine <3.0 <0.01
Isoleucine <3.0 <0.01
Methionine (Yeast) Methionine 44 0.016
Leucine <0.4 <0.001
: Valine <0.4 <0.001
Phenylalanine <0.4 <0.001
TaBie VIII

Methionyl adenylate and methionyl RNA formation tn various
fractions obtained during purtficaiton of methionyl
RNA synthetase from yeast

 

 

Methionyl | Methionyl
Enzyme fraction* fonntion | formation A/B X 10"
(A) (B)

pmoles/ myumoles/

mg/hour mp/hour
Crude extract....................- 0.61 0.23 2.6
Alcohol Fraction 2................ 14.8 5.2 2.8
Ammonium sulfate Fraction 1..... 23.2 8.0 2.9
Ammonium sulfate Fraction 2..... | 44.0 16.2 2.7
Alumina Cy gel eluate............ 55.7 19.7 2.8

 

* The various enzyme fractions were prepared as previously
described (35) and the specific enzyme activities for the formation
of methionyl adenylate and methionyl RNA were measured as
described under ‘‘Methods.”’

adenylate formation to that of leucyl RNA formation was 40
in crude extracts and 43 in the purified preparation (50-fold
purified). The analogous ratios with methionine in the crude
and purified preparations (75-fold purified) were 79 and 81, re-
spectively.

The failure to observe separation of amino acyl adenylate and
amino acyl RNA formation might also result (a) if the enzymes
involved fractionated identically in the procedures we have used,
June 1961 P. Berg, F. H. Bergmann, E. J.

TABLE IX

Comparison of rate of amino acyl adenylate and amino acyl RNA
formation under similar conditions

The rate of amino acyl RNA formation was measured as already
described, and the synthesis of amino acyl adenylates was deter-
mined by the amino acid-dependent ATP-PP;® exchange (26).
To compare the two rates, however, the ATP-PP,®? exchange re-
action was carried out under the same conditions used for amino
acyl RNA formation, except that unlabeled amino acid and 0.002
mM ATP and PP;® were added.

 

 

 

 

Amino acyt] Amino acy!

Enzyme focmtios formation A/B

(B)

pmoles/mg/hour
Leucyl RNA synthetase........... 120 2.8 43
Valyl RNA synthetase. ........... 574 26.4 22
Isoleucyl RNA synthetase......... 460 3.3 140

Methionyl RNA synthetase (£.

COW) cc ees 244 3.0 81
Methionyl RNA synthetase (yeast).| 22.1 0.007 3200

 

and (6) if the hypothetical enzyme catalyzing the transfer of the
amino acyl group to RNA were present in excess in both the
crude and subsequent enzyme fractions. ‘he first point can
only be answered by more extensive purification studies. The
second objection, however, is eliminated by the observation that
with each enzyme preparation it is the formation of the amino
acyl adenylate which is by far the faster reaction, ¢.e. the transfer
of the amino acyl group to the RNA is the rate-limiting step
(Table IX). Note that this difference in the rate of amino acyl
adneylate- and amino acyl RNA formation is of the order of
20- to 140-fold and in one case is about 3000 times. These data
imply that the transfer of the amino acyl moiety from the en-
zyme-amino acyl adenylate complex to the acceptor RNA is the
rate-limiting reaction. It should be pointed out that the slow
transfer of methionine by the yeast methionyl RNA synthetase
may be due to the use of the acceptor RNA from E. colt.

At present, our data are consistent with the hypothesis that a
single enzyme catalyzes both the formation of a specific enzyme-
amino acyl adenylate complex and the transfer of the amino acyl
group to the acceptor RNA,

DISCUSSION

The findings reported here and those presented recently by
other workers (16, 17) indicate strongly that the so-called “amino
acid-activating enzymes” are in essence amino acyl RNA syn-
thetases. Whereas the initial reaction between ATP, amino
acid, and a specific enzyme results in the formation of an enzyme-
bound amino acyl adenylate, in the presence of the appropriate
acceptor RNA chain, the amino acyl moicty is transferred to
the RNA and more specifically to the 2’- or 3’-hydroxyl group
of the terminal nucleotidyl ribose. A mechanism of this type
not only minimizes spontaneous destruction of the highly un-
stable free amino acyl adenylate under physiological conditions
(34, 36), but it also eliminates the requirement of additional
specific enzymes to form each amino acyl RNA derivative. In-
deed, it has recently been shown (37) that synthetic trypto-
phany] adenylate, in the presence of purified tryptophanyl RNA
synthetase, serves as tryptophan donor to amino acid acceptor
RNA. From a mechanistic view, the amino acyl RNA synthe-

Ofengand, and M. Dieckmann 1733

tases are analogous to the enzymes which catalyze the formation
of acyl-CoA derivatives (38-40), pantothenic acid (41), and
carnosine (42) in that there is a primary formation of an enzyme-
bound acyl adenylate and a subsequent transfer of the acyl
moiety to an acceptor molecule.

Recently Zillig et al. (43) reported that the yield of amino acyl
RNA is a function of the amount of amino acyl RNA synthetase
added. We have not observed this phenomenon in our studies.
Rather, only the initial rate of amino acyl RNA formation is
influenced by the amount of enzyme present. The final yield of
amino acyl RNA is, with sufficient time, independent of enzyme
concentration and depends entirely on the amount of acceptor
RNA present. In our early studies of valyl RNA synthesis, we
observed that the yield of valyl RNA did vary with the amount
of enzyme added. This, however, was found to be due to in-
activation of the enzyme during the course of the reaction, and
it could be circumvented by the addition of serum albumin to
the incubation mixture. Under these latter conditions, the en-
zyme continues to act until the acceptor RNA is saturated with
respect to valine.

The finding that amino acyl RNA synthesis is reversible is
surprising in light of the ester linkage between amino acid and
the acceptor RNA. The K., of 0.32 for valyl RNA synthesis |
and the values of 0.7 and 0.37 reported for threonyl RNA
synthesis (16, 17) indicate that the amino acyl moiety is main- |
tained at a high energy level. Whether this thermodynamic
activation of the amino acid is a consequence of an adjacent
hydroxyl group on the ribose or to some other structural feature
of the combination remains to be determined, —

An interesting aspect of the mechanism of amino acyl RNA
synthesis concerns the basis of the specificity in linking each
amino acid to the appropriate polynucleotide chain. This ques-
tion may be considered on the basis of the two reactions catalyzed
by the enzyme: in the first phase, the enzyme forms a specific
enzyme-amino acyl adenylate complex and in the second this
complex reacts with a specific acceptor RNA chain to form the
appropriate amino acyl RNA derivative. With respect to the
first phase of the reaction, it is clear from studies with the purified
amino acyl RNA synthetases that they exhibit a relatively high
degree of selectivity for a single naturally occurring amino acid.
The significance of the slight activity sometimes noted with other
amino acids is difficult to assess in the absence of more precise
data concerning the purity of the enzyme preparations and the
amino acid substrates (26). There are two exceptions, however,
which should be noted. The purified isoleucyl RNA synthetase
forms valyl adenylate as well as isoleucyl adenylate, and the
valyl RNA synthetase forms threonyl adenylate (26). In both
cases, the K, for the “unnatural” substrate is about 100-fold
higher than that for the “natural” one, so that with equal con-
centrations of the “natural” and “unnatural” amino acids, the
enzyme reacts almost exclusively with the ‘‘natural’”’ substrate.

In an analysis of the factors which control the transfer of the
amino acyl moiety to its specific acceptor RNA chain, several
aspects must be considered. First, we might ask, “What por-
tions of the synthetase-amino acyl adenylate complex function
in selecting the appropriate acceptor RNA chain?” Our data
suggest that both the amino acid and protein moieties function
in this selection. The fact that the enzyme-isoleucyl adenylate
complex transfers isoleucine to the isoleucine-specific RNA chain
but that the same enzyme in combination with valyl adenylate
does not transfer the valine to any acceptor RNA chain empha-
1734

sizes the role of the amino acid side chain. Similarly, the ob-
servation that different amounts of methionyl RNA are formed
when methionyl adenylate is linked to two different proteins
points to a specific function for the protein in the selection of
the correct RNA chain. There is no information at present con-
cerning the chemical structures of the RNA chains which allow
for the “recognition” between a specific enzyme-amino acyl
adenylate complex and its appropriate RNA chain. Clearly, the
terminal nucleotide, to which the amino acid is bound, cannot
account for this specificity since for each amino acid this unit is
adenylic acid (16, 24, 25). Whether the differentiation between
acceptor RNA chains relies on differences in nucleotide sequence,
configuration, or to some unknown factors remains to be deter-
mined. The indications that there may be heterogeneity
amongst RNA chains specific for a single amino acid may serve
to complicate the analysis of this problem.

SUMMARY

Purified enzymes from Lscherichia colt which form L-leucyl-,
L-valyl-, L-isoleucyl-, or L-methionyl adenylates also catalyze
the formation of the corresponding amino acyl ribonucleic acid
derivatives. Each amino acid is bound through its carboxyl
group to the terminal nucleotide (2’- or 3/-hydroxyl end) of
specific polynucleotide chains. The synthesis of amino acy] ribo-
nucleic acid derivatives is reversible, and in the case of L-valyl
ribonucleic acid formation the equilibrium constant is 0.32. In-
dications were obtained that the polynucleotide chains specific
for accepting L-methionine are heterogeneous.

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