THE JOURNAL OF BIOLOGTCAL CHEMISTRY
    Yol. 236, No. 6, June 1961
      Piinled in U.S.A.

The Enzymic Synthesis of Amino Acyl Derivatives
          of Ribonucleic Acid

II. THE PREPARATIOK OF LEUCYL-, VALYL-, IsomucYr,-, AND METHIOYYL

RIBONUCLEIC ACID SYNTHETASES FROM ESCHERICHIA COLI*

         FRED H. BERGM.4NNJ7 PAUL BERG,$ AND hl. DIECKMANN~

From the Department of Microbiology, Washiizgton Unicersity Xchool of Medicine, St. Louis 10, Missouri

            (Received for publication, November 8, 1960)

The enzymic synthesis of amino acyl ribonucleic acid deriva-
tives appears to take place in two steps, both of which are cata-
lyzed by a single enzyme (1-4).  The first step involves the
reaction of adenosine triphosphate, an amino acid, and an enzyme
specific for that amino acid, resulting in the formation of an en-
zyme-amino acyl adenylate complex and free inorganic pyro-
phosphate.  In the subsequent reaction, the amino acyl moiety
is transferred to the 2'- or 3'-hydroxyl group of a terminal
adenylic acid in a specific ribonucleic acid chain.  The present
paper describes the isolation and characterization of the en-
zymes that catalyze the formation of leucyl-, valyl-, isoleucyl-,
and methionyl ribonucleic acid.

EXPERIXIENTAL PROCEDURE

Escherichia coli strain B or ML30 was grown in a medium con-
taining 1.1 % KeHP04, 0.85% KH2POa,O.G% Difco yeast extract,
and 1% glucose.  The cells were grown with vigorous aeration
and harvested near the end of the period of esponcntial growth.
The cells were washed with a solution containing 0.5% NaCl and
0.5% KCI, recentrifuged, and stored at -15O.I  Cells stored for
several months in this way did not lose significant amounts of
enzymatic activity.

Both the DL- and L-forms of the amino acids used were obt,ained
from the California Foundation for Biochemical Research (CFP
grade), or from Nutritional Biochemicals Corporation.
ATP was purchased as the crystalline sodium salt from the
Sigma Chemical Company.  DEAE-cellulose, type 40, was pur-
chased from The Brov-n Company, and it was converted to the
hydroxyl form before equilibration with the appropriate buffers.
Superbrite glass beads, 200 p average diameter, were obtained
from the Minnesota Mining and Manufacturing Company, and
they were washed, before use with 1 N HC1 and then with water.

1'3*-labelcd sodium pyrophosphate was prepared by heating

* This investigation was supported by grant funds from the
National Institutes of Health of the Unitcd States Public 1-Iedth
Service.
t Postdoctoral Research Fellow of the National Institutes of
Health, United States Public Health Service; present address,
Department of Biochemistry, Brandeis University, Waltham,
Massachusetts.
1 Present address, Department of Biochemistry, Stanford Uni-
versity School of Medicine, Palo Alto, California.
1 We are deeply grateful to Dr. A. Hanson of the Grain Process-
ing Corporation for his help in obtaining E. coli cells grown in
this way.

NaHP3204 for 1 hour at 400" (5) and after separation from any
remaining P3I inorganic phosphate by chromatography on a
Dowex 1-C1- column (6), it was diluted with carrier sodium
pyrophosphate to a final specific activity of lo4 to lo5 c.p.m.
per pmole.  Protein determinations were carried out by the
method of Lowry et al. (7).

Paper chromatographic separation of isoleucine and valine was
carried out with a mixture of pyridine, iso-amyl alcohol, and H20
(35:35:30) (8).
The activity of each of the amino acyl RNA synthetases was
assayed by the amino acid-dependent eschange of ATP and PPi32
as described earlier (9).  Potassium fluoride (0.01 hi), which WI'RS
not inhibitory to the ATP-PP;32 reaction, was routinely added
to the reaction mixtures to inhibit any inorganic pyrophospha-
tase.  One unit of enzyme activity is equivalent to the incor-
poration of 1 pmole of PP,3* into ATP in 15 minutes.  As already
pointed out (9), the initial rate of incorporation of PYi3' into
ATP was equivalent to the initial rate of amino acyl adenylate
formation.  Fig. 1 shows that for each assay the rate of the
ATP-PPi32 exchange was proportional to thc amount of each
amino acyl RNA synthetase preparation used.  This propor-
tionality was observed in the range of 0.02 to 0.3 pmole of ATP32
formation in 15 minutes (0.02 to 0.3 unit).

RESULTS

Crude extracts of E. coli catalyze the formation of the amino
acyl adenylate derivatives of leucine, valine, isoleucine, and
methionine, as well as the format,ion of the corresponding amino
acyl RNA derivahives (10).  The same initia.1 fractionation pro-
cedures served for the purification of thc valyl-, isoleucyl-, and
lcueyl RNA synthetases, and eventual separation of these activ-
ities was achieved by chromatography on a DEAE-cellulose
column (11).  The methionyl RNA synthetase was obtained by
another procedure.

Isolation of Balyl-, Isoleucyl-, and LeuczJl RNA Synthetases-
Unless otherwise stated, all operations were carried out at 0-5".
A mixture of 100 g wet weight of E. coli cells, 100 ml of 0.025 IYI
Tris buffer, pH 8.0, and 300 g of glass beads (200 p) was stirred
in a stainless steel Waring Blendor run at about 90 volts.  If the
temperature of the mixture approached 5O, stirring was stopped
and the material was cooled in an ice bath to 0".  After a total
of 15 minutes of stirring, 300 ml of Tris buffer were added, and
the mixture was stirred at low speed for an addhional 2 to 3
minutes.  After thc beads had settled, the supernatant fluid was

1735

1736 Enzymic Synthesis of Amino Acyl RNA Derivatives. 11 Vol. 236, No. 6

Fro. 1. The rate of ATP-PPis* exchange as a function of the
amount of isoleucyl- (A), vaIyl- (B), leucyl- (C), and methionyl
RNA synthetases (D) added.  The reaction mixture contained,
in a volume of 1.0 ml, 100 prnoles of Tris buffer, p1-I 8.0; 5 pmoles
of hlgC12; 5 pmoles of potassiuni fluoride; 2 pmoles of ATP; 2
pinoles of P32-sodium pyrophosphate containing   t.o lo5 c.p.m.
per pmole; and 2pmoles of the appropriate amino acid.  The incu-
bation was carried out at 37" for 15 minutes and the amount of
radioactivity in tlie ATP was determined as previously described

(9).

decanted, and the beads were washed twice with 150 nil aliquots
of buffer.  The first supernatant fluid and washings wcre coni-
hined and centrifuged at 20,000 x g in a Serval1 SS-1 centrifuge
for 1 hour and the precipitate discarded.  The total volume of
extract was usually 600 to 700 ml.

For each 100 nil of cell extract, 5 ml of 1 M PIlnCls were added
with stirring, and after 20 minutes, the mixture was centrifuged
for 15 to 20 minutes as described above.  To every 100 ml of
the supcrnatant fluid were added 31 g of ammonium sulfate, and
aftcr 20 minutes, the mixture was centrifuged at 30,000 X g for
1 hour and the precipitate discarded.  To the supernntant fluid
mere added 12 g of ammonium sulfa.te, and after the mixture
was stirred for 20 minutes, it was centrifuged at 30,000 X g for
15 minutes. The precipitnte obtained from 100 g of cells was
dissolved in GO to 65 ml of 0.02 M phosphate buffer, pII 7.5, and
dialyzed for 5 hours against the same buffer.  The dialyzed
solution was clarified by centrifugation (ammonium sulfate frac-
tion 1).

To every 100 ml of nminoriiurn sulfate Fraction 1 were added
16 ml of 0.5 M potassium phosphate buffer, pH 7.5.  The solution
was incubated at 30" for 1$ hours, chilled to O", and then cen-
trifuged to remove any insoluble material.  By this procedure
most of the nuclcic acid in ammonium sulfate Fraction 1 is con-
verted to an acid-soluble form, facilitating the further purification
of the cnzymes.  The breakdown of the nucleic acids results from
the action of a ribonucleasc present in the ribosomal particles
(12).  To each volume of the supernatant fluid were added 0.4
volume of 0.05 M ATP, pH 7.1, 0.2 volume of 0.1 M L-valine, 0.2
volume of 0.1 M MgC12, 0.14 volume of 0.05 M KF, and 0.06
volume of 0.5 M K2HPOa.  The mixture was heated at 55" for
45 minutes, and after cooling to 0", the insoluble niaterial was
removed by cent,rifugation.  To each volunic of the heated su-
pernatant fluid, 1.6 volumes of ammonium sulfate solution,
snturated at 4", were added with stirring ovcr a 10-minute period.
The precipit,ate was removed by centrifugation at 30,000 X g
for 10 minutes and dissolved in 20 ml of 0.02 M phosphate buffer,
pH 7.5, containing 0.05 N 2-mercaptoethanol.  This solution
was dialyzed against the same buffer for 2 to 3 hours, centrifugcd,

and any insoluble material was discarded (ammonium sulfate
Fraction 2).
Although the heating procedure was designed to specifically
protect the valyl RNA synthetase, about 50y0 of the activity
with isoleucine and leucine remained after this treatment.  Be-
cause there a,ppeared to be considerable denaturation oE protein
and tlicreforc some purification of cadi activity, the procedure
was routinely used.

The dialyzed ammonium sulfate Fraction 2 was adsorbed to a
DEAE-cellulose column (3.8 cm2 x 16 cni) which had previously
been equilibrated with 0.02 M phosphate buffer, pH 7.5, contain-
ing 0.05 M 2-mercaptoethanol.  A linear gradient of decreasing
pH and increasing phosphate concentration was used to elute
the enzymes.  The nixing chamber contained 550 ml of 0.02 M
phosphate buffer, pH 7.5, whereas the reservoir contained 550
ml of 0.25 M phosphate buffer, pH 6.5; both solvents contained
0.05 M 2-rr,ercaptoetlianol.  Tlie flow rate was maintained at 50
to 60 in1 per hour and fractions of approsimately 25 ml were
collected.  The isoleucyl-, valyl-, and leucyl RN,4 synthetases
were eluted after about 400, 600, and 800 nil of buffer, respec-
tively, had passed through the column (Fig. 2).  The major
portion of cach peak was contained in 75 to 100 1x1.  The frac-
tions containing the major portion of each peak mere pooled and
the enzymes were concentrated by addition of solid ammonium
sulfate to 0.85 saturation.  After 15 minutes, the precipitat,e was
centrifuged at 30,000 x g for 1 hour and then dissolved in 2 to 3
ml of 0.02 M phosphate buffer, pH 7.5, containing    M reduced
glutathionc.  The individual cnzymes were dialyzed overnight
against about 100 volumes of the same buffer, and then against
fresh buffcr for another 3 to 4 hours.  Any precipitate which
formed during the dialysis was removed by centrifugation, and
the enzynics were stored at -15".

Isolation of Methionyl RNA Synthetase-Extract from 100 g of
E. coli was prepared as described above, and then dialyzed over-
night against 20 liters of 0.01 M Tris buffer, pII 8.0.  For each
volume of dialyzed extract, 0.9 volume of 0.05 M Tris buffer,
pH 7.5, and 0.1 volume of 2 M potassium phosphate buffer, pH
7.2, were added.  This mixture was incubated at 30" for 2.5 to 3
hours, and after it was chilled to O", the small amount of precipi-
tate was removed by centrifugation.  As previously pointed out,
this procedure facilitated the subsequent fractionation steps by
depolymerizing the nucleic acids present in the extract. To
each 100 ml of this solution were added 21.1 g of ammonium sul-

70

-

60

-

_I 50-

,

40-

?30-
t
? 20-

-

z

o

b- U

10-

250 500 750 1000

0

VOLUME,ML.

FIG. 2. Chromatographic separation of isoleucyl-, valyl- and
leucyl RNA synthetases on a DEAE-cellulose column.  See text
for complete details.

June 1961

Fractions

F. H. Bergmann, P. Berg, and M. Dieckmann

Volume

1737

23;:;

uds/
nts protein

                   TABLE I
Purijication oJ valyl-, isuleucyl-, and leucyl RNA synthetases Jrom E. coli

Total units'

11,400
6,850
G , 0GO
3,420
2,850

units/
nrg protein

1.3
3.4

lG


166

Protein
concen-
tration

Crude extract. ................
Ammonium sulfate 1.. .............
Ammonium sulfate 2 .............
DEAE-cellulose fractions?, ........
Valyl RhTA synthetase. ...........

nig/nrl

13.4
16.4
17.1

5.2
7.2
4.5

635
105
22

3.3

Val: ' RNA synthetase

Fraction

Dialyzed crude extract .....
Ammonium sulfate 1.. ......
.4mmonium sulfate 2.. ......
Cy gel eluate .............
Methioriyl RNA synthetase.

Total units'

     Protein
Volume concen-
     tration

__-

tnl mg fml

425    13.7
172    6.2
72    5.7
100     1.4

5 3.0

Specific
activity

Isoleucyl RNA synthetase I

Leucyl RNA synthetase

Total units'

16,600
11,550
5,320
3,770

3,120

Specific
activity

tmits/

mg protein
  2.7
  7.0

14

120

* Corrected for activity of isoleucyl RNA synthetase with valine.  (The true valine-dependent Al'P-PPiTp exchange a,ctivity is
? The actual recovery of each enzyme from the coliinin was: valyl RNA synthetase, 4860 units; isoleucyl RNA synthetase, 46SO
                 The values shown in the table represent the amounts in the most active fractions which

equal to the valine-dependent ATP-PPi32 exchange activity minus 0.55 of the isoleucine-dependent ATP-PPi32 exchange activity.)

units; leucyl IZN.4 synthctasc, 3875 units.
were pooled and used in the ammonium sulfate concentration step.

fate, and after stirring for 10 minutes thc solution was centri-
fuged.  To the supernatant fluid were added 6.3 g of ainmoniuni
sulfate, and aftcr 10 minutes, the precipitate was removed by
centrifugation and dissolved in about 20 ml of 0.02 M potassium
phosphate buffer, pH 7.2 ammonium sulfate Fraction 1).

To each 100 ml of ammonium sulfate Fraction 1, 18.3 g of
animonium sulfatc and then 6.3 nil of 1.8 M acetic acid were
added.  After about 5 minutes, the precipitate was removed by
centrifugation, and 4.1 g of ammonium sulfate were added to
the supernatant fluid; the mixture was again centrifuged, and
the precipitate was dissolved in 40 nil of 0.02 M potassium phos-
phate buffer, pH 6.5 (ammonium sulfate Fraction 2).

To each 100 ml of ammonium sulfate Fraction 2 were added
250 ml of cold water, and then 25 nil of alumina Cr gel (15 mg
dry weight per ml).  After 10 minutes, the mixture was ccntri-
fuged at 10,000 x g, the gel was washed with about 250 ml of
cold water, and the washings were discarded.  The enzymc was
eluted from the gel p-ith 150 ml of 0.1 M potassium phosphate
buffer, pH 7.0, and this fraction was dialyzed overnight against
50 to 100 volumes of 0.02 ai potassium phosphate buffer, pH 7.5
(Cy eluatc).

Of the dialyzed Cy eluate fraction, 100 in1 were adsorbed to a
DEAE-cellulose column (3.2 cni2 x 7 cm) which had previously
been equilibrated with 0.02 M potassium phosphate buffer, pH
7.5.  The enzyme was eluted with a linear gradient of increasing
ionic strength produced with a mixing chamber with 300 ml of
0.07 M potassium phosphate buffer, pH 7.0, and a reservoir of
300 ml of 0.2 M pot,assium phosphate buffer, pH 7.0.  The flow
rate was inaintaiiied at 50 to 60 in1 per hour, and 100 ml frac-
tions were collected.  The major portion of thc cnzyme was
eluted in about 75 ml (Fractions 17 to 25).  These frac.tions
were pooled, and the enzyme mas concentrated by precipitation
with ammonium sulfate as described for the other enzymes.  The
pellet was dissolved in 5 ml of 0.02 M phosphate buffer, p1-I 7.5;
any insoluble material mas removed by centrifugation.  The en-
zyme solution was stored at -15".

The data relative to the purification of the valyl-, isoleucyl-,
leucyl-, and methionyl RKA synthetases are suiiimarized in
Tables I arid 11.

Sta.bility of Purijied Enzyme Fractions-Preparations of the

most purified valyl- and isoleucyl RNA synthetases were rca-
sonably stable when kept frozen at -15" in the presence of
reduced glutathione   M) for periods as long as 3 to 4 months.
The loss in activity ranged froin 10 to 30%, and these prepara-
tions could not be react,ivated by the addition of 2-mercapto-
ethanol, reduced glutathione, or cysteine.  In the absence of
reduced glutathione, approximately 95 76 of the initial activity
was lost after 1 month, but such preparations could be reactivated
to about 50% of the initial level by the addition of 10-3 M 2-mer-
captoethanol to the assay mixture.  The purified preparations
of niethionyl RNA synthetase showed little or no loss in activit,y
on storage at -16", and no stimulation of the activity with
glutathione or 2-mereaptoethanol was ever observed.

In general, the purified leucyl RNA synthetase preparation was
the most labile on storage.  After several weeks at -15", there
mas a marked dependence of the activity on 2-mercsptoethanol
(or glutathione).  With some aged and completely inactive prep-
arations up to 90% of the activity could be restored by the addi-
tion of the 2-mercaptocthanol.  This stimulation of the activity
by 2-mercaptoethanol was found with Tris, glycylglycine, /3 ,/3-
dimethylglutarate, and imidazole buffers, but there was marked
inhibition by 2-mereaptoethanol when cacodylate buffer \vas
used.

Requirements for B 1'P-PPi32 Exchange Reaction-With each of
the purified enzymes there was 1 % or less of the activity of the.
complete system if either the amino acid, A@++, ATP, or the

              TABLE I1
Pur@cci,tion of methiany1 RNA synthetase from E. coli

Total
units

7245
3870
2304
1710
1363

Specific
activity

anils/nzg

protei,t
1.2
3.8
5.6
12.2
91

1738

L-Valine .._...., ,,....
D-Valine ... . . , . _. . . . . . .
L-Isoleucine . , . . . . . .
D-Isoleucine . . . . . . . . . . .
1,-Leucine. . . . . . . . . .
D-Leucine . . . . , . , . . . . . .
IJ-Methionine .. . . . .
r,-Threonine . . , . . . .

Enzymic Synthesis of Amino Acyl RNA Derivatives.

11

100
<1
1

< 1


<1
28

,

enzyme mas omitted.  The addition of 2-mercaptoethanol to
the reaction mixture usually had a stimulatory effect.  Leucyl
RNA formation showed the greatest effect being activated 3 to
5 times in thc presence of 2-mercaptoethanol.  In the case of
the valyl and isoleucyl RNA Synthetases, the activation was
between 1.5 and 3 times depending upon the age of the prepara-
tion.

Effect of pH and Type of Buffer on Rate of ATP-PPi32 Ex-
change-Although each of the isolated enzymes displayed little
variation in activity in the pH range of 7 to 9 there were marked
differences with various buffers.  For example, with the valyl
RNA synthetase, maximal activity with Tris or glycine buffers
was observed between pH 8.5 to 9.0, whereas with Cacodylate
or glycylglycine buffer, the maximal rate, which was about 10%
higher than those found with Tris and glycine buffer, was found
between pH 7.0 and 8.0.

In the case of the isoleucyl RNA synthetase, the activity was
masimnl at pH 8.5 (glycine or Tris buffers).  There was approxi-
mately 50% of thc activity at pH 9.5 (glycine buffer), whereas at
pH 6.6 (cacodylate buffer) there was 55% of the maximal ac-
tivity.

With the leucyl RKA synthetase there was little difference in
the activity in the pH range of 6 to 9 with Tris, glycine, glycyl-
glycine, or @, @-dimethylglutarate buffers.
The methionyl RSA synthetase had the same activity bc-
tween pH 6.0 and 8.8 and was approximately 40 and 30% as
active at pK 5.3 (cacodylate buffer) and pH 9.3 (glycine buffer),
respectively.
Determinuhion oj I<, Values for ATP und Amino AcidsThe
K,,, values for each of the amino acids were estimated by the
Linewaver-Burk (13) plot from data obtained in the standard
ATl'-PPiS2 exchange assay (Fig. 3).  These are: L-isoleucine,
5 x M; L-valine, 1 X M; L-leucine, 5.6 X M; and
mnethionine, 2.4 X hi.

The I<,,, values for ATP in the formation of va.ly1-, leucyl-,
and met,hionyI RNA under the conditions already described (2)
are 2.3 x 10-5 M, 1.3 x IO-` M, and 8.5 x   M, respectively.

Specificity of Isolated Amino Acyl RNA Synthetases in Formation
        of Amino Acyl Adenylates
(A) Nuturally Occurring Amino AcidsTable 111 shows the
results of experiments in which each of the isolated enzyme

5 10

15- 15-

200 600 1000

Vol. 236, No. 6

               TABLE 111
Specificity of valyl-, isoleucyl-, leucyl-, and melhionyl RNA
   synthstuses for amino acyl adenylate formation
The results have been expressed with the specifically activated

amino acid as 100 and all others relative to that figure, so that
results with differcnt preparations carried out at different times
could be tabulated.  Each amino acid was tested under the con-
ditions already described at a final concentration of L-amino acid
of 2 X     M.  The results with DL- or L-amino acids were the
same escept for certain instances noted in the test.

Amino acid tested* I %t

synthetase

Isoleucyl    Leucyl
RNA     RNA
synthetase  synthetasc

Methionyl
RNA
synthetase

44-56
(1
100
<1
2-5

5-10
<1

4


<1


100

<1
2
<1

<1


<1


2


100

<1

* Experiments not included in the table with the r>-isomers of
phenylalanine, tyrosine, tryptophan, lysine, serine, alanine, ar-
ginine, histidine, cysteine, and glycine with each of the enzymes
shown gave values less than 5% of that found with the appro-
priate amino acid.  L-Proline, L-glutamic, and  aspartic acids
were tested only with the methionyl RNA synthetase and found
to have less than 1% the activity of L-methionine.

preparations was tested for its ability to catalyzc the ATP-PPi32
exchange reaction in the presence of the different naturally occur-
ring amino acids.  Aside from the reaction of L-valine with the
isoleucyl RNA syntheta.se, and L-threonine with the valyl RNA
synthetase, both of which will be discussed subsequently, each
of the enzymes was found to show high specificity for a single
amino acid.  It should be pointed out that in certain instances
anomalous results were obtained with certain commercially ob-
tained samples of L-amino acids.  For example, several samples
of L-leucine gave significant amounts of exchange with the
methionyl RNA synthetase ranging from 10 to 50% of that
found with L-methionine, whereas other samples of the same
amino acid were inert. Similar observations were made with
samples of  isoleucine and  valine with the leucyl RNA syn-
thetase.  W'e believc that such anomalous results are due to
contamination of these L-amino acid preparations by other
L-amino acids.  Since the K, values for the amino acids by their
respective enzymes (see above) were relatively low and because
each amino acid preparation was routinely tested at a concen-
tration of 2 x   SI (with respect to the L-isomer), it is clear,
for examplc, that an impurity of 1% of L-methionine in the
L-leucine preparation or of L-leucine in the L-isoleucine and L-val-
ine preparations would be sufficient to account for a significant
exchange reaction.  Moreover, since the isotope exchange is not
accompanied by any net change in concentrations of the react-
ants, the trace impurity functions catalytically.  Support for
this conclusion comes from the fact that synthetically prepared
DL-amino acids were found to be uniformly inactive.  This was
not due to inhibition by the D-isomer since mixing equal amounts
of the o-amino acid with the active L-amino acid samples gave
no decrease in the rate of reaction.  These findings are being
stressed inasmuch as several investigators have tested the spec-

June 1961

30
0
100
15

F. H. Bergrnann, P. Berg, and M. Dieckmann

1739

ificity of these enzymes by Ihe ATP-PPi3? exchange reaction
(14, 15) with commercially obtained L-amino acid preparations.
(B) Evidence for Formation of L-Valyl Adenylate by L-Isoleucyl
  Synthetase-During the analysis of the enzyme fractions
eluted from the DEAE-cellulose column, two separate peaks of
valyl adenylate-forming activity were found (Fig. 2).  One of
these (Peak 1) was found to react with L-isoleucine as well as
L-valine.  Moreover, the &io of activity with isolcucinc and
valine was constant (1.72 to 1.82) in all fractions of the peak
(see Fig. 2).  The materkl in this peak accountcd for ovcr 85%
of the isoleucine-dependent ATP-PP,32 exchange activity of am-
monium sulfate Fraction 2 and about 30 % of thc valinc-dcpend-
ent activity.  The finding of two separable activities for valyl
adenylate formation and the constant ratio of activity with
isoleucine and valine in the first peak suggested the existence of
a single enzyme capable of forming both isoleucyl- and valyl
adenylate.  The following lines of evidence support this hy-

pothesis.

1. The activity observed in the prcscnce of equimolar but
saturating amounts of isoleucine and valine are not additive but
rather equal to the activity with isoleucine alone.  This would
be predicted on the basis of thc 100-fold difference in K,,, values
for the two amino acids as mentioned below.

2. The ratio of activities with isoleucine and valine remains
constant during inactivation and reactivation of the enzyme.
In one preparation in which 2-mercaptoethanol was omitted
from the eluting solutions, approximately SOY, of the activity
was lost whether tested with isoleucine or with valinc as sub-
strate.  Moreover, incubation of the partially inactivated en-
zyme preparation with 2-mcrcaptoethanol resulted in equal res-
toration of the two activities.

3. The possibility of contamination of the valine samples with
a trace of isoleucine as the basis for t.he observed activity with
valine can be eliminated.  With several different samples of
L-valine or m-valine, the ratio of the two activities (with isoleu-
cine and valine) did not vary significantly.  When a mixture of
L-isoleucine and L-valine was chromatographed with a solvent
which separates the two amino acids (8), the material recovered
from successive 0.5-cm strips cut perpendicularly to the line of
solvent flow gave two separate peaks of activity when tested in
licu of the amino acid in the standard assay system.  The posi-
tion of these two peaks corresponded to that for the isoleucine
and valine.

4. As pointed out in the previous paper (a), although this
enzyme produces both isoleucyl- and valyl adenylates, it cata-
lyzes the formation of only isoleucyl RNA.  Nevertheless, L-Val-
ine competitivcly inhibits the reaction linking L-isoleucine to
RNA, and the Ki for this inhibition is 3.8 x    M, whereas
thc K,, for valyl adenylate formation under the same conditions
is 3.9 x M.

These experiments suggest that whereas the isoleucyl RNA
synthetase can form both Lvalyl- and L-isoleucyl adenylates, the
enzyme differentiates between the two aniino acyl adenylate
derivatives in thc subsequent transfer of the amino acid residue
to RNA.
(C) Utilization of Bnalogues of Valine by Valyl RNA Synthe-
tase-The purified valyl RNA synthetase preparation shows
some activity in the ATP-PPia exchange reaction with amino
acid derivatives structurally related to valine (Table IV).  DL-
Threonine, which differs from DL-valine in the replacement of a
methyl group by an hydroxyl group, has a 100-fold greater K,

                 T.4BLE Iv
Activity of analogues of DL-valine with valyl RNA synthetase
All derivatives were tested under the conditions described un-
der "Experimentul Proccdure," cxcept that thc substrate con-

centrations were varied to determine the IC,,, values.

Substrate

oL-Valine .  . . . . . . . . . . . . . . . . , . . . . . . . . .
oL-Threoni ne. . . . , . . . . . , , . . , . .
~L-4llothreonine. . . . . . . .  . . . . . . . .   .
DL-ol-Aniino$-chlorobutyrate. . . . .  . . .
DL-Allo-a-amino-8-chlorobutyrate. . . .
uL-a-Aminobutyrate . . . . . , . . . . . . . . . . .
DL-u-Aminoisobutyrate . . . . . . . . . .  . . . .
oL-B,B-Dimethylcysteine. . . . . . . , . . . . .

K,*

'V

1 x 10-4
1.2 x lo-'

3.3 x 10-4
1 x 10-3
3.7 x 10-3

~~

* The K, values are calculated in each case for the ~-isuiiier.

(based on the L-isomer) than valine and the maximal rate is 30%
of that found with DL-valine.  DL-Allothreonine, in which the
hydroxyl group is of opposite configurat.ion to that of m-thrco-
nine, is inert. m-a-Amino-P-clilorobutyrate, in which the
chlorine atom has the same configuration as the hydroxyl group
of threonine, is at saturating conccntra.tions as active as DL-
valine, although the K, is slightly higher.  DL-Allo-a-amino-0-
chlorobutyrate, which corresponds to DL-dlothreonine, is only
15% as active as m-threonine.

It is interesting to note that the DL-a-arniiio-P-chlorobut):rtte
analogue of DL-threonine is a strong inhibitor of valine incorpo-
ration by animal cells in vitro whereas the ~~-allo-a-amino-p-
chlorobutyrate is only 2070 as active.2  Substitution of one of
the methyl groups of DL-valine by hydrogen, as in DL-a-amino-
butyrate, results in an increase of the K, and a decrease in the
V,,,. DL-a-hiniiioisobiityrate (norvaline) and DL-~ ,P-di-
niethylcysteine (penicillamine) are inert.  These data show that
the valyl RNA synthetase can differentiate between closely
related structures in the formation of t.he enzyme-amino acyl
adenylate complexes.  Further studies are required to evaluate
whether the ardogues which arc converted to the adenylate
derivatives are transferred to the valine-specific acceptor KNA
chains.

DISCUSSION

It is now generally accepted that the meclianism of the amino
acid-dependent exchange of ATP and PPP is explained by the
reversible formation of an enzyme-bound amino acyl adenylate.
To date, enzymes highly specific for the formation of L-methio-
nyl- (9), L-tryptophanyl- (15), L-tyrosyl- (16), L-alanyl- (17),
L-threonyl- (4), L-seryl- (18), D-alanyl- (19) , L-valyl-, L-isoleucyl-,
and L-leucyl adenylates have been isolated.  A closer inspection
of several of these enzymes revealed that they catalyze not only
the formation of a specific amino acyl adenylate but also the
synthesis of the corresponding amino acyl RNA derivative
(24, 17, 19, 20), and therefore may be considered as amino acyl
RNA synthetases.  Although each enzyme acts stoichiometri-
cally in the formation of the enzyme-amino :icy1 adenylate com-
plex, it appears to be regenerated during the subsequent syn-
thesis of the aniino acyl RNA compound (2).

tional Institutes of Health.
generous gift of the DL-amino-0-chlorobutyrate derivatives.

2 Private communication from Dr. M. Rabinovita of thc Na-
             We arc also grateful to him for the

1740 Bnzymic Synthesis of Amino Acyl RNA Derivatives. II Vol. 236, No. 6

The present work ha.s revealed a somewhat anomalous situa-
tion with rcspect to the isoleucyl- and valyl RNA synthetases.
In the former case, L-valine is utilized at slightly lcss than one-
half the rate of isoleucine, and in the latter, L-threonine reacts
at about onc-third the rate of L-valine. In both instances,
however, the K,, of the less active amino acid is about 100 times
higher than that of the "natural" substrate.  It seems reasonable
to suppose, therefore, that at conditions in vivo there is little
competition between amino acids for the appropriate enzyme.

Of further interest is the finding that the isoleucyl ltNA syn-
thetase will not form valyl RNA even though it makes the
enzyme-valyl adenylate (2). It is not yet clear, however,
whet,her such discrimination in the second reaction occurs with
other synthetases which show low level activity with "unnatu-
ral" amino acids and what the basis for this discriniination may
be.
It niiglit be pointed out that purified amino acyl RNA syn-
thctases provide a relatively simple and specific tool for measuring
the concentrations of a given L-amino acid in the presence of its
D-analogue or in the presence of a mixture of amino acid.  The
relatively low K, values for t'he amino acids in the ATP-PPP
exchange reaction (10-4 to 5  x 10-6 M) makes it possible to
measure amino acid concentrations in this range.  For exam-
ple, Stevens et al. (21), using the methionyl RNA synthetase
from yeast, have measured the biosynthesis of L-methionine by
liver extracts.

SUMMARY

Amino acyl ribonucleic acid synthetases, relatively specific for
either L-valine, L-leucine, L-isoleucine, or L-methionine, have
been purified from extracts of Escherichia coli.  The following
statemeots summarize some of the pertinent findings made with
the various Synthetase preparations.

1. The purified valyl-, isoleucyl-, and leucyl RNA synthe-
tases are relatively unstable in the absence of a sulfhydryl-
containing compound, whereas the methionyl ribonucleic acid
synthetase preparation shows no sulfhydryl requirement.
2. The IC, values of 1 X M (L-valine), 5 x 10-6 iv
(isoleucine), 5.6 X M (L-leucine), 2.4 X M (L-methio-

nine) were found with the respective enzymes in the formation
of the amino acyl adenylates.
3. Specificity studies show t,hat although the leucyl- and
methionyl ribonucleic acid synthetases are highly specific for a
single naturally occurring amino acid, isoleucyl ribonucleic acid
synthetase forms ~-valyt adenylate at about one half the rate
of L-isoleucyl adenylate formation, and valyl ribonucleic acid
synt,hetase utilizes threonine at about 30 :h the rate of valine.
Nevertheless, valine is not converted t.0 valyl ribonucleic acid by
the isoleucyl ribonucleic acid synthetase.

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