Eur. J. Biochem. 101,469-473 (1979)

Interaction of Arginine with the Ribosomal Peptidyl Transferase Centre Enrique PALACIAN and David VAZQUEZ Instituto de Bioquimica de Macromoleculas, Centro de Biologia Molecular Consejo Superior de Investigaciones Cientificas and Universidad Autonoma de Madrid (Received May 9, 1979)

Arginine inhibits the formation of acetylleucyl-puromycin from C(U)-A-C-C-A-LeuAc and puromycin (‘fragment reaction’), catalized by Escherichia coli and yeast ribosomes. From 18 different L-amino acids assayed, arginine was the most effective in producing inhibition (50% inhibition at 20 mM, with 1 mM puromycin). L-Argininamide and D-arginine gave about the same inhibition as L-arginine. The inhibition by L-arginine is competitive with respect to puromycin. The plot. of the slopes obtained in a Lineweaver and Burk representation versus [Arg]’, and the plot of l / v versus [Arg]’ at a fixed concentration of puromycin, are linear, which seems to indicate that two arginine molecules must interact at the puromycin binding site to produce inhibition. In addition to the ‘fragment reaction’, arginine inhibits the non-enzymatic binding of AcPhe-tRNA, C(U)-A-C-C-A-Leu and C(U)-A-C-C-A-LeuAc to ribosomes. However, it does not inhibit poly(U)-directed polyphenylalanine synthesis or the reaction of puromycin with AcPhe-tRNA previously bound to the peptidyl site. The results agree with arginine binding to the acceptor site, and with a sequential mechanism for the ‘fragment reaction’, puromycin binding first. reports the inhibitory effect of arginine on the ‘fragThe ‘fragment reaction’ assay, formation of acetylment reaction’ as well as its effects on binding to the leucyl-puromycin from C(U)-A-C-C-A-LeuAc and A and P sites. puromycin in the presence of 30% (v/v) ethanol and 70-S ribosomes or 50-S subunits, is a very useful assay for peptidyl transferase in spite of being quite unphysMATERIALS AND METHODS iological [l]. It allows the study of an isolated functional step of the complex ribosome activity using subMaterials strates of very small size as compared with the physiological ones. Two promising approaches to study Ribosomes from Escherichia coli MRE600 were peptidyl transferase are the use of the simplest riboprepared by grinding the cells with alumina, followed by differential centrifugation. Ribosomes were washed somal particles able to catalyze the reaction, and of three times with 1 mM NH4Cl, suspended in 5 mM the smallest possible ligands, model substrates and inhibitors. Puromycin and 2’(3’)-O-(N-formylmethio-Tris-HC1(pH 7.8), 20 mM magnesium acetate, 500 mM NH4Cl, 2 mM dithiothreitol, 0.5 mM EDTA and 50 ”/, ny1)adenosine 5’-phosphate are the smallest known substrates of peptidyl transferase [2]. However, in (v/v) glycerol, at a ribosomal concentration of 50 70 mg/ml, and kept at - 20 “C. Yeast ribosomes were transesterification and hydrolysis, which are also obtained from Saccharomyces cerevisiae haploid strain catalyzed by the peptidyl transferase active centre, the Y166 (a, his4, trp5, MA1) by grinding the cells with acceptor substrate can be as simple as ethanol and sea sand, differential centrifugation, and one-time water [l]. Some antibiotics inhibit the ‘fragment reacwashing with 0.5 M NH4C1 [5]. Yeast ribosomes were tion’ as well as the ‘fragment’ binding to either the A suspended in 100 mM Tris-HC1 (pH 7.4), 80 mM KCl, or P site, or both [3]. There is interaction between the 12.5 mM MgC12 and 1 mM dithiothreitol, at a ribotwo sites, since binding of antibiotics and deacylated some concentration of 100mg/ml, and kept at - 70 “C. tRNA to presumably one of the two sites produces Argininamide was synthesized from commercial stimulation of binding to the other site [3,4]. L-arginine methyl ester as previously described [6]. The inhibitors of the ‘fragment reaction’ studied The solutions of argininamide and those of the amino so far have been either antibiotics or structural analoacids were adjusted to pH 8.0. gues of portions of the natural substrates. This paper

470

Interaction of Arginine with the Ribosomal Peptidyl Transferase Centre

Pep tidy1 Transferase Assay Peptidyl transferase was estimated by the 'fragment reaction' assay, using C(U)-A-C-C-A-[3H]LeuAc and purimycin as substrates [7]. Unless otherwise indicated, the reaction took place at 0 "C for 24 min, and the reaction mixture contained 35 mM Tris-HC1 (pH 7.5 at room temperature), 12 mM magnesium acetate, 270 mM KC1, 6 mM 2-mercaptoethanol, 10- 20 nM C(U)-A-C-C-A-[3H]LeuAc (60 Ci/mmol), 1 mM puromycin and 30 % (v/v) methanol. This assay mixture has a pH close to 8.0 at 0 C. Binding to Ribosomes of Acetylphenylalanyl-tRNA and Synthesis of Acetylphenylulunyl-puromycin

I

I

20

40 [Arginine] (mM)

I

I

60

80

The binding of acetylphenylalanyl-tRNA to E. coli Fig. 1. Inhibition of peptidyl trunsferose ,from E. coli rihosomc,.~hy ribosomes was performed in 20 mM Tris-HC1 (pH 7.8), ctiffeerent concentrations of urginine. The reaction mixtures for the 12 mM magnesium acetate, 70 mM NH4C1, 4 m M fragment reaction assay contained the arginine concentrations dithiothreitol, 50 pg/ml poly-(U), 52 nM A ~ - [ ~ H ] p h e - indicated. In the absence of arginine, the amount of acetylleucylpuromycin synthesized was 0.32 fmol min-' pmol ribosomes-' tRNA ( 3 Ci/mmol) and 211 nM ribosomes. After incubation at 30 "C for 20 min, the reaction mixtures were filtered through Whatman GF/C filters, and the Table 1 . Ejfects of arginine on the non-enzymatic binding of acetylradioactivity was measured. To estimate acetylphenylphenylalanyl-tRNA to the ribosomes and the formation of acetylalanyl-puromycin synthesis, at the end of the prephenylalunyl-puromycin ceding incubation 1 mM puromycin was added, and Experimental conditions are indicated in Materials and Methods. Arginine was added prior to the first incubation to determine its incubation at 30 "C was continued for another 10 min. effect on binding, and at the time of puromycin, after the first incuAcetylphenylalanyl-puromycin was extracted with bation, to estimate its effect on AcPhe-puromycin formation ethyl acetate, and the radioactivity was measured. Addition

Binding to Ribosomes of the Acetyluted and Non-acetylated 'Fragments'

AcPhe-tRNA binding

AcPhe-puromycin formation

pmol ~

Binding was performed as previously described [8]. The assay mixture contained 30 mM Tris-HC1 (pH 7.9, 12 mM magnesium acetate, 270 mM KCI, 40 mM NH4C1, 6 mM 2-mercaptoethanol, 50 % (v/v) ethanol, 2.0- 2.7 nM C(U)-A-C-C-A-[3H]Leu or C(U)-A-CC-A-[3H]LeuAc (60 Ci/mmol), and 4.5 mg/ml ribosomes.

RESULTS Inhibition of Peptidyl Transferase by Arginine The amino acid arginine inhibits peptidyl transferase, assayed by the fragment reaction, of E. coli (Fig. 1) and yeast (Fig. 3 ) ribosomes. Fig. 1 shows inhibition of peptidyl transferase catalyzed by E. coli ribosomes at different concentrations of arginine. In the range of concentrations used in Fig. 1, arginine does not inhibit poly(U)-directed polyphenylalanine synthesis. On the contrary, it produces stimulation that reaches 100 % increase in activity at 50 mM arginine (not shown). At this concentration, arginine does not inhibit the reaction of puromycin with acetylphenylalanyl-tRNA previously bound to the P site

None 50 mM arginine

0.34 0.21

~~

0.29 0.36

(Table 1). However, it inhibits the non-enzymatic binding of acetylphenylalanyl-tRNA to the ribosome (Table 1). The dependence of the extent of inhibition of the fragment reaction on arginine concentration is not that expected for inhibition produced by binding of one molecule of inhibitor per active centre (Fig. 1). The plot of the reciprocal of reaction rate versus the square of arginine concentration produces a straight line (Fig. 1, insert), suggesting that two molecules of arginine must interact with the ribosome to produce inhibition. Similar results were obtained with 50-S subunits (not shown). The inhibitory effect of arginine was reversible upon dilution. E. coli ribosomes incubated for 30 min at 0 "C with 50 mM arginine, and diluted in the fragment reaction assay mixture to give an arginine concentration of 5 mM, had 88% of the activity of the untreated control, which is a value very close to that obtained for 5 mM arginine without preincubation at a higher concentration of inhibitor (Fig. 1).

E. Palacian and D. Vizquez

471 I

0

5

10

1 /[ Puromycin] (mM-')

0

20 4 0 0 500 1000 1503 [Arginine] (mM) [Arginine]* (rnM2)

Fig. 2. Inhibition by arginine of peptidyl tranSferasefrom E. coli ribosomes ui dqferent concentrations ofpuromycin. (A) Peptidyl transferase activity was determined at the concentrations of puromycin indicated. The concentrations of arginine were as follow: 0 (O), 10 mM (a), 20 mM (v),30 mM (B), and 40 mM (A). u is expressed as fmol acetylleucyl-puromycin formed min-' pmol ribosomes-'. (B) Replots of the slopes from (A) versus arginine concentration and versus the square of arginine concentration

15

: 10

0

50 100 I / [Puromycin ] (rnM-')

1/ [C (U)-A-C-C-A- Leu Ac] (nM-')

Fig. 3. Inhibition by arginine of'peptidyl transfferusr, f r o myeast ribo.somes at different concentrations ofpuromycin. Peptidyl transferase was determined in the presence (A) and absence (0) of 30mM arginine, at the concentrations of puromycin indicated. ti is expressed as fmol acetylleucyl-puromycin formed min-' pmol ribosomes-'

Fig. 4. Inhibition by arginine of peptidyl trirtisjrrase from E. coli ribosomes at different concentrations of C ( Uj-A-C-C-A-LeuAc. Peptidyl transferase was determined in the presence (A)and absence ( 0 ) of 20 mM arginine, at the concentrations of C(U)-A-C-C-ALeuAc indicated. u is expressed as fmol acetylleucyl-puromycin formed min-' pmol ribosomes-'

The formation of acetylleucyl-puromycin by E. coli ribosomes in the presence and absence of arginine was proportional to time for the first 25 min of reaction, the percentage inhibition produced by arginine being thus independent of the reaction time.

than that of E. coli ribosomes. Yeast ribosomes are less susceptible to arginine inhibition than those from E. coli, because to obtain about the same degree of inhibition it was necessary to double the arginine concentration with yeast ribosomes (Fig. 2 and 3). With E. coli ribosomes the plot of the slopes obtained in a Lineweaver and Burk representation versus the square of arginine concentration is linear (Fig. 2B), which seems to indicate that two arginine molecules interact at the puromycin binding site, in agreement with the results shown in Fig. 1. On the other hand, the inhibition by arginine of peptidyl transferase is non-competitive with respect to C(U)-A-C-C-A-LeuAc (Fig. 4).

Competition of Arginine with Puromycin The inhibition by arginine of peptidyl transferase is competitive with respect to puromycin with ribosomes from E. coli (Fig. 2) and yeast (Fig. 3). To see inhibition with yeast ribosomes, the concentrations of puromycin have to be lowered, since the apparent K,,, for puromycin was found a factor of 10 lower

Interaction of Arginine with the Ribosomal Peptidyl Transferase Centre

472

-

Table 2. Efjrects of different amino acids on the fragment reaction assay with E. coli ribosomes Addition of the following amino acids at a concentration of 30 mM gave 90-110% of the activity of the control: glycine, L-alanine, L-serine, L-leucine, L-asparagine, L-threonine, L-valine, L-proline, ~-isoleucine, L-glutamine and L-cysteine. The controls without additions synthesized 0.35 (Expt 1) and 0.41 (Expt 2) fmol acetylleucyl-puromycin min-' pmol ribosomes-' Expt

Addition

Fragment reaction (relative activity)

1

none L-arginine (30 mM) L-lysine (30 mM) L-phenylalanine (30 mM) L-histidine (30 mM) L-aspartic acid (30 mM) L-methionine (30 mM) L-glutamic acid (30 mM)

100 34 69 72 74 79 87 89

none L-arginine (50 mM) D-arginine (50 mM) L-argininamide (50 mM)

100 18 24 39

2

Specificity of Inhibition

The effects of different amino acids, in addition to arginine, on peptidyl transferase activity were also determined. Of the amino acids tested arginine was by far the most effective (Table 2). Lysine, phenylalanine, histidine and aspartic acid produced significant inhibition, while methionine, glutamic acid, glycine, alanine, serine, leucine, asparagine, threonine, valine, proline, isoleucine, glutamine and cysteine had little or no effect. On the other hand, D-arginine and L-argininamide inhibited peptidyl transferase to about the same extent as L-arginine, which was the arginine form used in the rest of the experiments (Table 2).

I

100

50

2 01

I

0

~

100

50 [Arginine] (mM)

Fig. 5. Effect of arginine on the binding of C(UJ-A-C-C-A-Leu ( 0 ) and of C(U)-A-C-C-A-LeuAc (A) to the E. coli ribosomes. In the absence of arginine the ribosomes bound 52 % and 30 % of the added non-acetylated and acetylated fragments respectively. The results are expressed as relative values in order to compare the inhibition of binding of the two fragments

Table 3. Inactivation of E. coli ribosomes by arginine Ribosomes (7 mg/ml) were preincubated for 8 days at 0- 5 "C with arginine at the concentrations indicated in 70 mM Tris-HC1(pH 7.5 at room temperature), 16 mM magnesium acetate, 50 mM NKCI, 0.5 mM dithiothreitol, 0.4 mM EDTA and 5 :/, (v/v) glycerol. Prior to the fragment reaction assay, aliquots of the preparations were heated at 30 "C for 17 min. Activity was assayed in aliquots of the preparations, with addition of arginine in some cases to give the final concentrations shown in the table. The activity of the ribosomes preincubated and assayed in the absence of arginine and not heated (relative activity 100) was 0.14 fmol acetylleucyl-puromycin formed min-' pmol ribosomes-' ~

~~

Arginine concentration ~

Fragment reaction (relative activity)

~~

preincubation

Effect of' Arginine on the Binding of the Acetylated and the Non-acetylated Fragment to Ribosomes

mM

In addition to peptidyl transferase and the binding of acetylphenylalanyl-tRNA, arginine inhibits the binding of C(U)-A-C-C-A-Leu and C(U)-A-C-C-ALeuAc to ribosomes (Fig.5). The binding of both fragments was inhibited in a similar degree.

0 0 50 50 100 100

assay

without previous heating

after heating

0 30 10 30 20 30

100 40 39 18

204 158

-

I 6

-

63 -

Inactivation of Ribosomes by Arginine

Besides its inhibitory effect, arginine inactivates E. coli ribosomes, and this inactivation can be partially reversed by heating at 30 "C. Table 3 shows the peptidyl transferase activity of ribosomes preincubated with arginine for 8 days and assayed at different arginine concentrations. The preparations preincubated with 50 and 100 mM arginine, and assayed at arginine concentrations of 10 and 20 mM respectively,

have activities much lower with respect to the control than those expected from the arginine concentrations in the assay mixtures (Fig. 1). When the preparations preincubated with arginine, as well as the control, were assayed in the presence of the same arginine concentration (30 mM), the same differences were observed : the preparations preincubated with arginine had activities much lower than the control. This inacti-

E. Palacian and D. Vazquez

vating effect of arginine was partially reversed by incubation at 30°C prior to assay. Although, even the control was notably reactivated by heating, the reactivation was proportionally much higher in the preparations preincubated with arginine. While the activity of the control was double upon heating, those of the preparations preincubated with arginine were 4 and 9 times higher than before heating.

DISCUSSION Arginine interacts directly or indirectly with the peptidyl transferase centre of the ribosome, as indicated by arginine inhibition of the fragment reaction and of the binding to the ribosomes of the acetylated and non-acetylated fragments, as well as that of acetylphenylalanyl-tRNA. The competitive inhibition with respect to puromycin of the fragment reaction is most directly explained as arginine binding to the puromycin site. This is also supported by the arginine inhibition of non-acetylated fragment binding to the A site. However, arginine also inhibits binding of acetylated fragment and acetylphenylalanyl-tRNA to the P site, while no competition with respect to the acetylated fragment was found in the inhibition of the fragment reaction. On the other hand, kinetic studies of the fragment reaction indicate that the mechanism is sequential [9]. In the physiological elongation cycle, the donor substrate is bound prior to acceptor binding, but this order of addition need not necessarily agree with that of the fragment reaction. Independent binding of the acetylated fragment and puromycin to the ribosome has been taken as indication of random addition of these substrates to the ribosomes during fragment reaction catalysis [9]. However, the separate binding of substrates might not be kinetically significant. Our results agree with this possibility. If puromycin binds first to the ribosome, the subsequent binding of the acetylated fragment should not be affected by arginine, since apparently arginine produces inhibition by binding to the A site, and this site is already occupied by puromycin. On the other hand, in the absence of puromycin, binding of arginine to the A site would inhibit the binding of the acetylated fragment to the P site. In the case of reaction of acetylphenylalanyl-tRNA with puromycin, when arginine is added the P site is already occupied by acetylphenylalanyl-tRNA, and this substrate bound to the P site might prevent the binding of arginine to the A site, thus avoiding the inhibitory effect of arginine, in agreement with the obtained results. Like sparsomycin and some aminonucleoside antibiotics, arginine inhibits peptide bond formation by

473

both prokaryotic and eukaryotic ribosomes [3]. The lack of inhibitory effect on polypeptide polymerization and on acetylphenylalanyl-puromycin synthesis is not surprising, since many antibiotics that inhibit the fragment reaction have no effect on peptide bond formation in intact cells or on the puromycin reaction with native peptidyl-tRNA using isolated polyribosomes [3]. If one molecule of arginine were bound to the A site in the correct position it might react with the acetylated fragment and acetylleucyl-arginine would be formed. We have tried to detect the formation of this compound as well as the one that would be formed with argininamide, an analogue of arginine that has the carboxyl group of arginine blocked, thus avoiding its effect on the reactivity of the cc-amino group. The results obtained were negative. The inhibitory effect of D-arginine as well as the binding of two molecules of arginine per ribosome to produce inhibition seem to indicate that arginine inhibition is not produced by binding at the same place where the aminoacyl moiety of the natural substrate is located on the peptidyl transferase centre. Since the 3’-terminal oligonucleotide of tRNA probably binds to a nucleotide sequence of 23-S rRNA [lo, 111, arginine might cause its inhibitory effect by interacting with the negatively charged backbone of this nucleotide sequence, and in this way inhibiting the binding of non-acetylated fragment or puromycin. This work was supported by an institutional grant to the Centro de Biologia Molecular liom Cotnisicin del Descuento Complemmtclrio (Insiituto Nacionul de Previsihn) and a personal grant from Essex Laboratories.

REFERENCES 1. Harris, R. J . Sr Pestka, S. (1977) in Molecular Mechunisms of Protein Biosynthesis (Weissbach, H. Sr Pestka, S.. eds) pp. 413-442, Academic Press, New York. 3. Ct-mi, J., Rychlik, I., Krayevsky, A. A. Sr Gottikh, B. P. (1973) FERS Let/. 37, 188-191. 3. Vazquez, D. (1979) Inhibitors of Protein Biosynthesis, SpringerVerlag, Berlin. 4. Ulbrich, B., Mertens, G. Sr Nierhaus, K. H. (1978) Arch. Biochem. Biophys. 190, 149-154. 5. Battaner, E. Sr Vazquez, D. (1971) Biochim. Biuphys. Acta, 254, 316- 330. 6. Dirk, K . Sr Spiith, H. (1935) Hoppe-Seyler’s Z. Pkysiol. Chem. 237, 124-130. 7. Monro, R. E. (1971) Methods Enzymol. 20, 412-481. 8. Celma, M. L., Monro, R. E. Sr Vazquez, D. (1970) FEBS Letl. 6 , 273 - 271. 9. Fernindez-Mufioz, R. Sr Vazquez, D. (1973) Mol. B i d . Rep. 1. 75 - 79. 10. Harris, R. J., Greenwell, P. & Symons, R. H. (1973) Biochem. Biophys. Res. Commun. 55, 117- 124. 11. Greenwell, P., Harris, R. J. Sr Symons, R. H. (1974) Eur. J . Biochem. 49, 539-554.

I?. Palacian and D. Vazquez, Institute de Bioquimica de Macromoleculas, Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas y Universidad Autonoma de Madrid, Facultad de Ciencias, Universidad Autonoma de Madrid, Canto Blanco, Madrid-34, Spain