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Eur. J. Biochem.

68, 1 - 3 (1976)

Indolmycin Inhibits Prokaryotic Tryptophanyl-tRNA Ligase


Rolf G. WERNER, Lance F. THORPE, Wolfgang REUTER, and Knud H. NIERHAUS

Dr. Karl Thomae GmbH, Biberach an der Riss,


and Max-Planck-Institut fur Molekulare Genetik, Abteilung Wittmann, Berlin-Dahlem
(Received April 9/June 4, 1976)

Indolmycin specifically prevents the formation of tryptophanyl-tRNA in a prokaryotic system


in vifvo using Eschevichia coli enzymes. However, the drug has little effect in an eukaryotic system
in vim (rat liver enzymes). Analysis of the type of inhibition revealed that indolmycin competes with
tryptophan as a pure competitive inhibitor of prokaryotic tryptophanyl-tRNA ligase.
Most of the antibiotics inhibiting protein synthesis
interfere directly with prokaryotic ribosomal functions.
A small number of antibiotics act as inhibitors of
steps taking place prior to translation (for review see
[l]).For example, the drug borrelidin prevents the
formation of threonyl-tRNA in some bacterial species

PI.

In this paper we show that indolmycin specifically


inhibits the tryptophanyl-tRNA ligase from Escherichia coli whereas the corresponding ligase from rat
liver is affected much less.
MATERIALS AND METHODS
Muter ials

ATP and tRNA from E. coli and calf liver were


obtained from Boehringer, Mannheim ;the radioactive
amino acids were obtained from New England Nuclear; Instagel scintillation liquid from Packard ; the
filters from Sartorius ;the total synthesis of indolmycin
followed described procedures [3 - 61; all other chemicals were from Merck, Darmstadt. The 300000xg
supernatant from rat liver was a generous gift from
Dr F. Grummt (Max-Planck-Institut fur Biochemie,
Munchen-Martinsried). The preparation of the 1SO 000
x g supernatant from E. coli has been described
previously [7].
Methods

Formation of aminoacyl-tRNA in the prokaryotic


system was performed in 100-p1 reaction mixture
containing 300 pg tRNA from E. coli, 6 p1 150000 x g
supernatant from E. coli and the following concentrations of other components: 50 mM Tris . HCl, pH 7.8,
70 mM KC1, 10 mM acetate, 4 mM 2-mercaptoethanol, 5 mM ATP and about 0.7-1 nM [3H]trypto~~

Enzyme. Tryptophanyl-tRNA ligase (EC 6.1.1.2)

phan (specific activity: 0.6 Ci/mol; or when indicated


an equivalent amount of another radioactive amino
acid). After incubation for 15 min at 30 "C the reaction was stopped by adding 2 ml of a precooled 7 %
(w/v) solution of trichloroacetic acid at 0 "C. The
precipitate was collected on Sartorius filters (regenerated cellulose, pore size 0.6 pm, cat. no. SM 116 0s).
The filters were washed twice with 7 % trichloroacetic
acid, dried and counted after adding Instagel scintillation liquid.
Aminoacyl-tRNA formation in the eukaryotic
system was performed under identical conditions
except that tRNA derived from calf liver and enzymes
from rat liver (8 pl 300000 x g supernatant per assay)
were used.

RESULTS AND DISCUSSION


The enzymic formation of [3H]tryptophanyl-tRNA
was optimized using the 1SO 000 x g supernatant from
E. coli (see Fig. 1A- D). The conditions chosen for
routine analysis (as described in Materials and
Methods) are indicated with an arrow in Fig. 1.
This prokaryotic system was used to test the effect
of indolmycin on the formation of 12 different aminoacyl-tRNAs. We selected the amino acids alanine,
isoleucine, phenylalanine, tyrosine, cysteine, methionine, glutamic acid, glutamine, arginine, histidine,
threonine and tryptophan, i.e. at least one example
from each of the different amino acid classes. Fig. 2A
demonstrates that indolmycin specifically prevents
the formation of tryptophanyl-tRNA; SO % inhibition
is found at an indolmycin concentration of about
30 pM with complete inhibition at 300 pM, whereas
concentrations of the drug as high as 10 mM do not
affect the formation of the other aminoacyl-tRNAs.
Indolmycin only causes slight inhibition of tryptophanyl-tRNA formation in an eukaryotic system

Action of Indolmycin

-t/---I;
50

' 0

20

<

40 60 80 100
KCI concentration [ m M I

100

200

300

4M)

500

per assay [ k g ]

tRNA

5 mM

2 ' 0
P

=l-

2
4
6
8
ATP concentration [rnM]

10

5300 enzymes [rat liver] per assay [PI]

5
10
15
20
5150 enzymes [ E . c o l i ] per assay [PI]

10

20
30 40 50
Incubation time [min]

60

Fig. 1. Optimization of the a . ~ . ~ a ~ vhf oe racriviiy of['HH]try~~tophaty:l-tRNA


ligase. The parameter varied is given on the abscissa in each case,
with the other conditions as described in Materials and Methods. (A- D) Prokaryotic system (with enzyme fraction and tRNA derived from
E. coli); (E) eukaryotic system (with tRNA from calf-liver); (F) kinetics ofthe optimized prokaryotic system, (0-0)
37 "C, (M
30)
"C.
The arrows indicate the conditions chosen for the optimized system and used in subsequent assays. S150 enzymes ( E . coli) and S300 enzymes
(rat-liver) are the 150000 x g supernatant from E. coli and the 300000 x g supernatant from rat liver, respectively

using tRNA from calf liver and 300000 x g supernatant from rat liver (see Fig. 2B). 10% inhibition is
seen at an indolmycin concentration of 30 pM, and
only 30% at 10 mM. The possible breakdown of
indolmycin by enzymes present in the crude rat liver
extract was tested by the following experiment.
Indolmycin was incubated in the presence of rat liver
extract at 30 "C for 60 min and the indolmycin activity
was examined before and after incubation. Similarly
indolmycin was incubated alone at 30 "C for 60 min
and the activity tested before and after incubation.
Activity was determined in vivo by measuring the
growth inhibition of Staphylococcus uureus. Incubation of the drug with and without rat liver extract did
not affect the indolmycin activity indicating that
indolmycin is not inactivated during incubation in
presence of rat liver extract. A hybrid system composed
of calf liver tRNA and 150 000 x g supernatant from
E. coli is inhibited even more strongly than the prokaryotic system (Fig. 2C). Thus, the inhibition of
indolmycin is related to the synthetase and not to the
tRNA fraction. Most likely, indolmycin competes
with tryptophan on the respective synthetase.
The last .experiment was designed to analyze the
type of inhibition (see [S]). The tryptophanyl-tRNA

formation was measured with increasing tryptophan


concentrations at different indolmycin concentrations and also in the absence of the drug. The reaction
was stopped after 3 min, and the data obtained were
plotted on a Lineweaver-Burk diagram (see Fig. 3).
The lines obtained from indolmycin competition
experiments ( i = 30 pM and 80 pM, respectively)
intersect the ordinate at the same point as the line
obtained from experiments without indolmycin (i= 0)
indicating that indolmycin inhibits the tryptophanyltRNA formation by increasing the apparent K,
whereas I/ is not affected. Therefore, indolmycin is a
competitive inhibitor. Furthermore, when 1/71 was
plotted against indolmycin concentration, straight
lines were obtained (not shown) indicating a pure
competitive action of the inhibitor. This conclusion
is supported by the finding that sufficiently high
concentrations of indolmycin depress the tryptophanyl-tRNA formation to mere background values
(see Fig. 2A). Thus, indolmycin acts as a pure competitive inhibitor of tryptophan binding to the respective
ligase. In view of the structural relationship between
tryptophan and indolmycin (see formula in Fig. 4),
it is not surprising that they compete for one and the
same binding site. With the data from the Lineweaver-

R . G. Werner, L. F. Thorpe, W. Reuter, and K. H. Nierhaus

In

1I [Tryptophan] (nM-' )

Fig. 3. Linewuvrr- Burk plot uf r r j p i o p h n j / - / R IVA ,furmnr Lori in


rhc presence of indohycin concenirations of 30 und 80 ph4. The
reaction time was 3 min; at the substrate concentration used, the
velocity during this incubation time corresponds to the initial
velocity

50t 1
4
0.001

0.01

0.1

10

Indolmycin concentration [mMl


Fig. 2. ~ n ~ ~ ~ of'
i / iiw
j o n
formation of uniinoucyL2RNAs. (A) Prokaryotic system; (B) eukaryotic system; (C) inhibition of the
formation of tryptophanyl-tRNA in a hybrid system using tRNA
from calf liver and 150000 x g supernatant (40 pl per assay) from
E. coli. (@-@)
Trp-tRNA formed; (0----O) 12 other aminoacyl-tRNAs formed (see Results and Discussion)

Burk diagram the inhibition constant (Ki) of indolmycin was calculated, for both concentrations to have
a value for K , = 8 - 9 x
M.
In conclusion, indolmycin specifically inhibits
prokaryotic tryptophanyl-tRNA ligase whereas the
corresponding eukaryotic ligase is only slightly affected. The effect of indolmycin on mitochondria (penetration into mitochondria, inhibition of the mitochondrial aminoacylation) was not analysed. It may
turn out that the drug can be used in vivo as a bacteriospecific agent, even if it inhibits mitochondria1 aminoacylation in vitro, if it does not penetrate into the mitochondria.
We thank Drs H . G. Wittmann and R. Brimacombe for advice
and helpful discussion.

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-

R. G. Werner*, L. F. Thorpe, and W . Reuter, Dr. Karl Thomae GmbH, D-7950 Biberach an der Riss,
Federal Republic of Germany

K . H. Nierhaus, Max-Planck-Institut fur Molekulare Genetik, Abteilung Wittmann,


Ihnestrasse 63/73, D-1000 Berlin (West) 33-Dahlem

* T o whom correspondence should be addressed.