14
Eur. J. Biochem. 97, 481 -494 (1979) Yeast Phenylalanyl-tRNA Synthetase Affinity and Photoaffinity Labelling of the Stereospecific Binding Sites Mireille BALTZINCER, Franco FASIOLO, and Pierre REMY Institut de Biologie Moleculaire et Cellulaire du Centre National de la Recherche Scientifique, Strasbourg (Received December 4, 1978/March 12, 1979) The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl-tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The tRNAPh' was cross-linked to the enzyme by non-specific ultraviolet irradi- ation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4-thiouridine (s"U) residues introduced in the tRNA molecule, or by Schiffs base formation between periodate-oxidized tRNAPh' (tRNA;:') and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation. The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA. The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the 0 subunit (M, 63000). The phenylalanine residue, either free or joined to the adenylate, could be cross-linked with equal efficiency to either type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits. The Schiffs base formation between tRNA:,he and the enzyme shows the existence of a lysyl group close to the binding site for the 3'-terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast phenylalanyl-tRNA synthetase with pyridoxal phosphate and the 2',3'-dialdehyde derivative of ATP, oATP. Yeast phenylalanyl-tRNA synthetase (hl, 260000), a tetramer composed of two a subunits (hi, 72000) and two p subunits (Ail, 63000), has been shown by Fasiolo et al. to be a functional dimer, two molecules of each ligand being bound per molecule of enzyme [l]. Both types of subunit appear to be required for the expression of enzymatic activity, since the isolated subunits have not yet been found to be active. Further- more, the tetrameric structure seems to be essential for the enzyme activity, as upon renaturation of a mixture of equimolecular amounts of separated M and /I subunits, activity is only found associated with material having a molecular weight of 260000 [2] (and Raffin and Remy unpublished results). ~l,hi.c,i,irr/ro,i.s. oATP, the 2',3'-dialdehyde derivative of ATP: tRNA::', periodate-oxidized tRNAPhe; tRNAEt:r,,,, periodate-oxi- dized tRNAPhr, reduced with NaBH4; [s4U]tRNAPhe, tRNAPh' in which some uridine residues have been replaced by 4-thiouridine. Enzjmcs. Yeast phenylalanyl-tRNA synthetase (EC 6.1.1.20); yeast tRNA nucleotidyl transferase (EC 2.7.7.25); T4 polynucleo- tide kinase (EC 2.7.1.78); ribonuclease TI (EC 3.1.4.8); pancreatic ribonuclease (EC 3.1.4.22); yeast pyrophosphatase (EC 3.6.1.1.); calf intestine phosphomonoesterase (EC 3.1.3.1). Previously studies of Fasiolo et al. [2] by proteo- lytic modification of phenylalanyl-tRNA synthetase suggested that the subunit was responsible for most of the interactions with tRNAPhe, since the binding of this ligand was lost after specific cleavage of and conversely the binding of tRNAPhe to phenylalanyl- tRNA synthetase strongly protected 6 subunit against mild tryptic hydrolysis. So far, no direct evidence has been obtained con- cerning the localisation of the specific binding sites. This paper wiIl report the results obtained by affinity and photoaffinity labelling of the accepting sites spe- cific for the different ligands of the enzyme: tRNAPh', ATP and amino acid, either in free form, joined to the adenylate, or linked to the tRNA. MATERIALS AND METHODS Enzymes Yeast phenylalanyl-tRXA synthetase was prepared by the procedure previously described [3]. Yeast tRNA nucleotidyl transferase was a gift from Dr Rether and

Yeast Phenylalanyl-tRNA Synthetase : Affinity and Photoaffinity Labelling of the Stereospecific Binding Sites

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Page 1: Yeast Phenylalanyl-tRNA Synthetase : Affinity and Photoaffinity Labelling of the Stereospecific Binding Sites

Eur. J. Biochem. 97, 481 -494 (1979)

Yeast Phenylalanyl-tRNA Synthetase Affinity and Photoaffinity Labelling of the Stereospecific Binding Sites

Mireille BALTZINCER, Franco FASIOLO, and Pierre REMY

Institut de Biologie Moleculaire et Cellulaire du Centre National de la Recherche Scientifique, Strasbourg

(Received December 4, 1978/March 12, 1979)

The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl-tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The tRNAPh' was cross-linked to the enzyme by non-specific ultraviolet irradi- ation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4-thiouridine (s"U) residues introduced in the tRNA molecule, or by Schiffs base formation between periodate-oxidized tRNAPh' (tRNA;:') and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation.

The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA.

The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the 0 subunit ( M , 63000). The phenylalanine residue, either free or joined to the adenylate, could be cross-linked with equal efficiency to either type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits.

The Schiffs base formation between tRNA:,he and the enzyme shows the existence of a lysyl group close to the binding site for the 3'-terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast phenylalanyl-tRNA synthetase with pyridoxal phosphate and the 2',3'-dialdehyde derivative of ATP, oATP.

Yeast phenylalanyl-tRNA synthetase (h l , 260000), a tetramer composed of two a subunits (hi, 72000) and two p subunits (Ail, 63000), has been shown by Fasiolo et al. to be a functional dimer, two molecules of each ligand being bound per molecule of enzyme [l]. Both types of subunit appear to be required for the expression of enzymatic activity, since the isolated subunits have not yet been found to be active. Further- more, the tetrameric structure seems to be essential for the enzyme activity, as upon renaturation of a mixture of equimolecular amounts of separated M and /I subunits, activity is only found associated with material having a molecular weight of 260000 [2] (and Raffin and Remy unpublished results).

~ l , h i . c , i , i r r / r o , i . s . oATP, the 2',3'-dialdehyde derivative of ATP: tRNA::', periodate-oxidized tRNAPhe; tRNAEt:r,,,, periodate-oxi- dized tRNAPhr, reduced with NaBH4; [s4U]tRNAPhe, tRNAPh' in which some uridine residues have been replaced by 4-thiouridine.

Enzjmcs. Yeast phenylalanyl-tRNA synthetase (EC 6.1.1.20); yeast tRNA nucleotidyl transferase (EC 2.7.7.25); T4 polynucleo- tide kinase (EC 2.7.1.78); ribonuclease TI (EC 3.1.4.8); pancreatic ribonuclease (EC 3.1.4.22); yeast pyrophosphatase (EC 3.6.1.1.); calf intestine phosphomonoesterase (EC 3.1.3.1).

Previously studies of Fasiolo et al. [2] by proteo- lytic modification of phenylalanyl-tRNA synthetase suggested that the subunit was responsible for most of the interactions with tRNAPhe, since the binding of this ligand was lost after specific cleavage of and conversely the binding of tRNAPhe to phenylalanyl- tRNA synthetase strongly protected 6 subunit against mild tryptic hydrolysis.

So far, no direct evidence has been obtained con- cerning the localisation of the specific binding sites. This paper wiIl report the results obtained by affinity and photoaffinity labelling of the accepting sites spe- cific for the different ligands of the enzyme: tRNAPh', ATP and amino acid, either in free form, joined to the adenylate, or linked to the tRNA.

MATERIALS AND METHODS

Enzymes

Yeast phenylalanyl-tRXA synthetase was prepared by the procedure previously described [3]. Yeast tRNA nucleotidyl transferase was a gift from Dr Rether and

Page 2: Yeast Phenylalanyl-tRNA Synthetase : Affinity and Photoaffinity Labelling of the Stereospecific Binding Sites

482 Yeast Phenylalanyl-tRNA Synthetase

was prepared as described in [4]. T4 polynucleotide kinase was a gift from Dr Keith and was prepared according to [5,6]. Ribonuclease T1 and pancreatic ribonuclease were purchased from Calbiochem (Los Angeles, U.S.A.). Yeast pyrophosphatase was ob- tained from Sigma (St Louis, U.S.A.) and calf in- testine phosphomonoesterase from Worthington.

Chemicals

Radioactive chemicals [3H2]0, [14C]phenylalanine and [3H]phenylalanine were purchased from the Com- missariat a 1'Energie Atomique (Saclay, France), [3H]- ATP and [14C]ATP from the Radiochemical Center, Amersham. [Y-~~P]ATP was synthesized by G. Keith according to the method of Glynn and Chappell [7].

All other chemicals were of the best available purity and were obtained from Merck (Darmstadt), Prolabo (Paris) or Sigma (St Louis). The acetone used in the sensitized photo-cross-linking experiments was ultraviolet grade solvent and was purchased from Merck (Darmstadt). Yeast total tRNA was obtained from Boehringer.

Preparation of Modified tRNAPh'

Yeast tRNAPhe was purified from total tRNA by counter-current distribution, as described by Dir- heimer and Ebel [8]. The accepting capacity was usually higher than 1500 pmol of phenylalanine/A260 unit.

To prepare yeast tRNAPhe-C-C-[3H]A, the 3'-ter- minal adenosine of tRNAPhe was split off by the chemical procedure described by Uziel and Khym 191. After elimination of the 3'-terminal phosphate ac- cording to Walker and Rajbhandary [lo], the tRNA was incubated with yeast tRNA nucleotidyl transferase in the presence of t3H]ATP, under the conditions de- scribed by Sprinzl et al. Ill]. The specific activities usually ranged around 100 counts x min-' x pmol-'.

Alternatively, purified tRNAPhe was also labelled on the ribose moiety of the 3'-terminal adenosine by periodic oxidation followed by reduction with NaB- [3H4] under the following conditions : tRNA:? (160 pM) was dissolved in 200 mM ammonium bi- carbonate pH 9.0 at 0°C and added to the tritiated borohydride (5 mCi, 10 - 26 Ci/mmol) and kept 15 min at 0°C in a well ventilated hood. A second reduction was performed with unlabelled sodium borohydride (130 mM) under the same conditions. The excess of borohydride was then destroyed by addition of acetic acid up to pH4.0 in the hood. The tRNA solution was then extensively dialysed against distilled water to eliminate all exchangeable protons. The specific activities obtained ranged between 1000 and 4000 counts x min-' x pmol-' according to the specific activity of borohydride.

To prepare yeast tRNAPhe-C-C-[3H]A,,, tRNAPhe labelled on the 3'-terminal adenosine was submitted to periodic oxidation under the following conditions: the tRNA was dissolved in 100mM acetate buffer pH 5.0 at a concentration of 160 pM; sodium meta- periodate was added at a final concentration of 10 mM and the oxidation was performed for 30 min at room temperature, in the dark, Reaction was stopped by addition of a large excess of glycerol and the tRNA was exhaustively dialysed against distilled water.

To obtain purine-labelled tRNAPhe, purified tRNAPhe was tritiated by isotopic exchange using [3H2]0, under conditions similar to those described by Shelton and Clark [12]. The labelling is strictly limited to C-8 of the purine residues. The specific activities obtained ranged from 100 to 400 counts x min-' x pmol-' depending upon the specific ac- tivity of the tritiated water used (usually 1 Ci/ml).

For yeast [s4U]tRNAPhe, thiolation was performed by Lapidot et al. (Jerusalem). The tRNA (120 pM) was dissolved in 150 mM phosphate buffer pH 7.2 containing 20 mM MgC12. The reaction was carried out during 100 h under pressurized H2S. Excess H2S was then removed under vacuum and the tRNA was recovered by ethanol precipitation. The precipitate was redissolved in water and dialysed extensively against 1 mM MgC12.

Preparation of Radioactive Aminoacyl-tRNA

Native tRNAPhe and [s4U]tRNAPhe were amino- acylated using ['4C]phenylalanine or [3H]phenylala- nine, in the following conditions: the tRNA at a con- centration of 80 pM was dissolved in 25 mM Tris- HCI buffer pH7.5, 10mM ATP, 15mM MgC12, 2 mM reduced glutathione, 0.2 mM labelled phenyl- alanine and 1 pM enzyme. After 20 min of incubation at 37"C, the reaction was stopped by addition of sodium acetate buffer pH 4.5 to a final concentration of 100 mM. The protein was eliminated by phenol extraction and the acylated tRNA was freed from small ligands by chromatography on DEAE-Sephadex A25 equilibrated with 100 mM sodium acetate buffer pH 4.5. The elution was performed with a linear con- centration gradient of NaCl (0.2 to 1 M) in the same buffer. The acylated tRNA was recovered by ethanol precipitation, dissolved in 1 mM acetate buffer pH 4.5 and exhaustively dialysed against the same buffer.

Ultraviolet Irradiations

The apparatus used for ultraviolet irradiations was bought from Cunow (Paris) and consists of a 2500-W mercuryjxenon lamp followed by a quartz predisper- sion prism and a Schoeffel grating monochromator (model GM 250) giving a bandwidth of 3 nm with a l-mm exit slit. Quartz lenses were used to focus the

Page 3: Yeast Phenylalanyl-tRNA Synthetase : Affinity and Photoaffinity Labelling of the Stereospecific Binding Sites

M. Baltzinger, F. Fasiolo, and P. Remy

Table 1. Output power in the focal plane of the irradiation bench

Wavelength Output power measured by

the thermopile actinometry

nm quanta/min

248 1 .o x 1 O l 8 6.5 x 101'

335 1.0 x lols 8.0 x 1017 313 2.5 x 10l8 2.5 x 10l8

light on 10 x4-mm quartz cell sitting in a thermo- stated holder. During irradiation the temperature was maintained at 4 "C. Usually the irradiations were per- formed at wavelengths corresponding to mercury lines, where the output presents important maxima. The output power in the focal plane was measured either with a thermopile (Kipp and Zonen, Delft, Holland) or by actinometry, according to Leighton and Forbes [13]. Very similar results were obtained, as illustrated in Table 1.

Direct Photo- Cross- Linking Experiments

Native tRNAPhe was cross-linked to phenylalanyl- tRNA synthetase using two different ultraviolet irra- diations: a non-specific irradiation at 248 nm and a specific irradiation in the wye base absorption band at 313 nm (with a bandwidth of 3 nm).

The irradiations were carried out in the following conditions. 1 ml of 20 mM sodium phosphate buffer pH 7.2, 10 mM EDTA containing 4 nmol of [3H]- tRNA:xh_qed or [p~r ine* -~H] tRNA~~" and 3.8 nmol of phenylalanyl-tRNA synthetase was irradiated at 4 "C. Aliquots were withdrawn at given time intervals and the formation of the covalent bond was followed by nitrocellulose disc filtration under dissociating con- ditions (0.5 M KCI in the same buffer). After washing and drying, the radioactivity was counted in 5 ml of scintillation mixture.

[14C]Phe-[s4U]tRNAPhe was cross-linked to phe- nylalanyl-tRNA synthetase by irradiation at 335 nm under the following conditions. I ml of 50 mM sodium acetate buffer pH 6, 10 mM MgCL containing 2- 4 nmol of enzyme and 0.5 - 1 .O nmol of tRNA was irradiated at 4 "C. At given time intervals, aliquots were withdrawn. The amount of covalent complex formed and residual enzymatic activity were measured.

Phenylalanine was cross-linked to phenylalanyl- tRNA synthetase, either as a free amino acid, or joined in the adenylate complex or bound to the tRNA, upon irradiation at 248 nm. For the free amino acid and the aminoacyl-tRNA, the reaction mixture contained : 50 mM acetate buffer pH 6.0, 10 mM MgC12, 1.2 pM phenylalanyl-tRNA synthetase, and either free [3H]- phenylalanine (50 pM to 5 mM) or Phe-tRNAPhe

50 mM KCI, 0.1 mM EDTA, 5.4 pM isolated enzyme- adenylate complex (stoichiometry 1.5 mol phenyl- alanine/mol enzyme). As above, aliquots were taken at given time intervals and assayed for covalent com- plex formation and enzyme inactivation.

Sensitized Photo-Crosslinking Experiments

Sensitized photo-crosslinking of ATP to phenyl- alanyl-tRNA synthetase was performed in the fol- lowing conditions. 0.1-ml reaction mixtures containing 20 mM sodium phosphate buffer pH 7.5, 37 pM phenylalanyl-tRNA synthetase, 500 pM [14C]ATP (125 Ci/mol), 5 % acetone were irradiated at 313 nm at 4°C. Aliquots were withdrawn and assayed for covalent complex formation and enzyme inactivation.

Affinity Labelling of Phenylalanyl-tRNA Synthetase

C3H]tRNA::" was reacted with phenylalanyl-t RNA synthetase at room temperature in a mixture con- taining 25 mM phosphate buffer pH 8.2, 5.4 pM [3H]tRNA::e (specific activity 120 counts x min-' x pmol- I), 2.7 pM phenylalanyl-tRNA synthetase. The final volume was 0.5 ml. At given time intervals, 50-p1 aliquots were withdrawn and diluted in 500 pl of 100 mM phosphate buffer pH 8.2, and immediately mixed with 3 pl of an NaBH4 solution (0.5 M solution in 10 mM sodium hydroxide). The reduction was allowed to proceed for 30 min at 0°C; the aliquots were brought to 0.5 M sodium chloride prior to filtra- tion on nitrocellulose membranes to determine the amount of covalent complex formed.

Reaction of Phenylalanyl-t RNA Synthetase with Pyridoxal.5'- Phosphate

1 pM enzyme was allowed to react with 1 mM pyridoxal 5'-phosphate at 25 "C in 100 mM phosphate buffer pH 7.2, 5 mM 2-mercaptoethanol (except when other conditions are specified). At given time intervals aliquots were taken and assayed for enzyme inactiva- tion either directly or after reduction by sodium boro- hydride as above.

Analysis of the Covalent Labelling Pattern

In the case of tRNA-enzyme covalent complexes, the tRNA moiety was first digested either with TI or pancreatic ribonuclease and the enzyme subunits were then separated by sodium dodecylsulphate/polyacryl- amide gel electrophoresis to study the label distribu- tion among the constitutive subunits. When needed, the bound oligonucleotides were post-labelled using

Page 4: Yeast Phenylalanyl-tRNA Synthetase : Affinity and Photoaffinity Labelling of the Stereospecific Binding Sites

484 Yeast Phenylalanyl-tRNA Synthetase

[p3'P]ATP and T4 polynucleotide kinase. The fol- lowing techniques were used :

TI and Pancreatic Ribonuclease Treatment of the tRNA- Phenylalanyl-tRhrA Synthetuse Covalent Complexes

The covalent complexes were usually separated from unbound tRNA by Sephadex G-100 filtration under dissociating conditions. Excluded material was pooled. When needed, the buffer could be changed by several centrifugations and washes in membrane cones (type Amicon Centriflo C F 50, Amicon Corp. Lexing- ton, U.S.A.). The hydrolyses were performed in 50 mM Tris-HC1 buffer pH 8.0, 0.1 mM diisopropylfluoro- phosphate, in order to prevent proteolytic cleavage of the enzyme during the incubation. When volatile buffers were required, the hydrolysis was performed in 70 mM ammonium bicarbonate pH 8.0 or 2- 5 '%, pyridine brought to pH 8.0 with acetic acid. The in- cubation was carried out at 37 "C for 3 h with 300 units of TI ribonuclease or 100 pg of pancreatic ribonuclease/ mg tRNA.

Post-labelling of the Bound Oligonucleotides

In some instances, the bound pancreatic oligo- nucleotides were post-labelled using [ Y - ~ ~ P I A T P and T4 polynucleotide kinase, as described by Simsek et al. [14].

Separation of Modified Phenylalanyl-tRhTA Synthetase Subunits

The constitutive subunits of phenylalanyl-tRNA synthetase were separated by sodium dodecylsulphate/ polyacrylamide gel electrophoresis, either on disc or on slab gels, according to the procedure of Laemmli [I 51. Protein bands were visualized by Coomassie blue staining (2 g Coomassie blue in 1 I 50% methanol, 7.5 % acetic acid), the destaining being performed with 50% methanol, 7.5% acetic acid. The slab gels were dried according to Maize1 [I61 and submitted to auto- radiography.

Determination of Enzymatic Activity

Conditions for the ATP-PP: exchange and the aminoacylation reaction of tRNAPhe were described previously [l , 2 ] .

RESULTS AND DISCUSSION

Covalent Binding of tRNAot:red to Phenylalanyl-tRNA Synthetase upon Irradiation at 248 nm

Fig. 1 shows the extent of covalent bond forma- tion as a function of time, analysed by filtration on

- g 12 N L a,

- 2 8 9 . - 0 E I

'0 3 6 9 12 15 18 21 Time (min)

Fig. 1. Covulent incorporution in yenst phaiiylu~uni~l-fRR'A synthelrrse qf tRNA""-C-C-['H]A"~-~',~ upon ir-rudiuiion a/ 248 nm. 3.8 pM phenylalanyl-tRNA synthetase, 4 p M [3H]tRNA:::rc, (4800 counts x min-' x pmol-I). (O--O) Covalent incorporation of tRNA:t:rc,, (0-----0) inhibition of the acylation activity of yeast phenylalanyl- tRNA synthetase

nitrocellulose membranes. It can be seen that tRNAPhe is incorporated almost linearly during the first 20 min of irradiation, the extent of binding approaching 12 (mol tRNA/mol enzyme). As shown, the inactivation of the acylation activity of phenylalanyl-tRNA syn- thetase is slightly more rapid than the covalent bond formation. This is not surprising since thiol groups are known to be very important for the acylation activity of phenylalanyl-tRNA synthetase 11 71 and these groups are also particularly sensitive to side reactions occurring during the irradiation (i.e. attack by radicals). But it must be emphasized that the rate of inactivation remains of the same order as the rate of covalent bond formation. Furthermore, it was shown in the valyl-tRNA synthetase system from yeast that the inactivation of the acylation activity of the enzyme was not correlated to a modification of the binding sites for tRNA since the irradiated enzyme always bound the tRNA with the same efficiency (M. Renaud, personal communication). It is thus highly probable that the higher rate of enzyme in- activation mostly reflects side modification of im- portant amino acids, without affecting the specificity of tRNA binding.

The irradiated reaction mixture was then digested with pancreatic ribonuclease as described above and the hydrolysate was fractionated by molecular sieving on a Sephadex G-50 column. Fig. 2 shows that the enzyme, appearing at the void volume, still contains the radioactive modified terminal adenosine. This result clearly shows that the modified terminal ade- nosine was covalently linked to the enzyme with good efficiency.

Page 5: Yeast Phenylalanyl-tRNA Synthetase : Affinity and Photoaffinity Labelling of the Stereospecific Binding Sites

M. Baltzinger, F. Fasiolo, and P. Remy 485

In order to study more easily the distribution of the covalent bonds formed between enzyme and tRNA, among the constitutive subunits of phenylalanyl- tRNA synthetase, the oligonucleotides remaining bound to the enzyme after pancreatic ribonuclease digestion and Sephadex filtration were post-labelled using Iy-32P]ATP and T4 polynucleotide kinase. The constitutive subunits of phenylalanyl-tRNA synthe-

. . . . . . . . . .

Vase were then separated by sodium dodecylsulphate/ polyacrylamide gel electrophoresis. Fig. 3 shows that only the p subunit ( M , 63000) of the enzyme was labelled, suggesting that most of the interactions be- tween tRNA and phenylalanyl-tRNA synthetase take place on this subunit.

Covalent Binding of Native tRNAPhe to Phenylulunyl-tRNA Synthetuse upon Irradjution in the Wye Buse Absorption Band

Fig. 4 shows the yield of covalent complex formed as a function of time. The semi-log plot (insert) is linear indicating that the incorporation is a pseudo- monomolecular reaction, which demonstrates that the reversible enzyme-tRNA complex has to be formed before the covalent incorporation may occur. The covalent binding yield is rather low, since after 60 min only 3 % of the binding sites specific for tRNA are modified. This incomplete joining can be attributed to the inactivation of the chromophoric group of tRNA, since as shown on Fig. 5 the addition of fresh tRNA results in a further incorporation. It must be emphasized that the inhibition of the acylation activity of phenylalanyl-tRNA synthetase appears to be stoi- chiometric to the amount of tRNA bound, since a 20% inactivation is observed for a 17.5% covalent binding yield.

In order to identify the subunit(s) of phenylalanyl- tRNA synthetase bound to the tRNA, the reaction mixture was brought to 7 M urea and loaded on a DEAE-cellulose column equilibrated in 150 mM phos- phate buffer pH 7.2,7 M urea. Under these conditions, free protein was not bound to the ion exchanger and

"0 2 4 6 8 10 12 14 16 18 20- Fraction number

Fig. 2. isolation by Sephndex G-50 filtration of yeast phenylulanyi- tRNA synthetase covotently labelled by terminal [3H]udenrxine of yenst i R h ~ A ~ , ~ ~ r , , ufter pancreatic ribonuclease treatment of the irradiated complex. Column size: 2 3 x 0 , s cm; buffer: 30 mM Tris-HC1 pH 8.0; fraction volume 1.1 ml. (-0) Absorbance at 280 nm; (e--e) absorbance at 260 nm; (A-----A) radioactivity

B

--a - - B

Fig. 3. identification of the subunit of phenylalunyi-tRNA syniherase involved in the covalent incorporation of tRhrA:,::',,, upon irradiution ar 248 nm and of' [s4U/tRhrAPh' during irradiution at 335 nm. (A) Staining of the sodium dodecylsulphate/polyacrylamide gel with Coomassie blue. (B) Autoradiography of the electrophoregram. (1) Native enzyme, (2) irradiation of the complex at 335 nm, (3) irradiation of the complex at 248 nm. The fast-migrating band identified as 8' is a partial proteolysis compound derived from a subunit, as previously shown [2]

Page 6: Yeast Phenylalanyl-tRNA Synthetase : Affinity and Photoaffinity Labelling of the Stereospecific Binding Sites

486 Yeast Phenyialanyl-tRNA Synthetase

Time (min)

Fig. 4. Covalent incorporation of [p~rine~-~H]tRh'A"'' ' ' into yeast phenylulanyl-tRh'rl synthetase upon irradiation in the wye base ab- sorption band at 313 nm. 5.2 pM phenylalanyl-tRNA synthetase. 11 pM [3H]tRNAPh' (200 counts x min-' x pmol-*). Insert: semi- log plot of the difference A I between incorporation at infinite time and incorporation at time t (arbitrary units) as a function of irradiation time

is washed out with the same buffer. The elution with 1 M NaCl in the same buffer allowed the recovery of free tRNA and covalent tRNA-protein complexe(s). The wye base of tRNAPhe was then split off by acidic treatment of the material recovered upon sodium chloride elution, according to Thiebe and Zachau [IS]. The subunit(s) liberated upon wye base excision was(were) then identified by sodium dodecylsulphate/ polyacrylamide gel electrophoresis, a standard enzyme being run at the same time. Table 2 summarizes the results obtained in three different experiments. In all experiments, the p subunit of phenylalanyl-tRNA synthetase ( M , 63000) was identified as the major component. A small amount of protein material having an electrophoretic mobility intermediate be- tween those of a and p subunit was present in two experiments. This extra band is probably due to an incomplete excision of the wye base upon acidic treat- ment, leaving some subunits crosslinked to a tRNAPhe molecule. Indeed, by sodium dodecylsulphate/poly- acrylamide gel electrophoresis of the irradiated mix- ture without acidic treatment, it was observed that the radioactive covalent tRNA-protein complex mi- grated between a and subunits. In one experiment a small amount of a subunit was also found (28% of the protein recovered). This contamination may arise either from an incomplete wash out of the a subunit

Fig. 5. Ejject of' multiple additions of' fRhrAph' upon covalent in- corporation of the latter in phenylalunyl-tRh'A synthetase during irradiation in the wye base absorption band. Initial conditions: 5.2 pM phenylalanyl-tRNA synthetase, 11 pM [3H]tRNAPhe (200 counts x min-' x pmol-I). At times indicated by the arrows, the same amount of fresh tRNA was added to the incubation mixture

Table 2. Identijicution of the subunit oJphmnylalany1-tRNA synthetase linked to tRNA"' upon irradiation in the wye base absorption band The relative mobilities on sodium dodecylsulphate polyacrylamide gels of u and subunits and of the subunit linked to tRNAPhe upon irradiation at 313 nm were measured. The numbers between brackets in the last column correspond to the percentage of each component in the analyzed sample

Expt Relative mobilities of subunits

U B linked to tRNAPh'

1 0.27 0.32 0.31 (P, 100%)

2 0.24 0.31 0.22 (u, 28%) 0.25 (23%) 0.28 (B, 49%)

0.34 ( B , =.90%) 3 0.28 0.34 0.31 (trace)

from the DEAE-cellulose column or from some un- specific binding of tRNA upon irradiation. Never- theless, the main target of the labelling appears to be the p subunit of the phenylalanyl-tRNA synthetase. This result shows that the anticodon loop of tRNAPhe mostly or even exclusively interacts with the p subunit.

Covalent Binding of [ l4 C ] Phe-(s4 U ] t R NAPhe to Phenylulanyl-tRNA Synthetase upon Irradiation at 335 nm

The thiolated tRNAPhe used in this study, [s4U]- tRNAPhe had between five and six modified uridine residues per molecule of tRNA. This modified tRNA

Page 7: Yeast Phenylalanyl-tRNA Synthetase : Affinity and Photoaffinity Labelling of the Stereospecific Binding Sites

M. Baltzinger, F. Fasiolo, and P. Remy

was found to be homogenous by polyacrylamide gel electrophoresis, the amino acid-accepting capacity was 880 pmol/&o unit. The binding of [s4U]tRNAPhe to phenylalanyl-tRNA synthetase was investigated by filtration of a mixture of 2 pM phenylalanyl-tRNA synthetase and 2.2 pM [s4U]tRNAPhe on a Sephadex column, in ionic conditions favouring the formation of the specific complex.

A single peak of complex (half-life > 2 h) was eluted from the column, without any free tRNA peak, indicating that the affinity for the modified tRNA was either unchanged or only slightly decreased, as com- pared to native tRNAPhe. The thiolated tRNA can therefore be a useful tool for investigation of recog- nition regions between the enzyme and the nucleic acid. Indeed, the drawback of cross-linking experi- ments using ultraviolet irradiation around 260 nm is the absence of correlation between covalent bond formation and enzyme inactivation. Upon irradiation at 335 nm of the free enzyme, no inactivation could be detected even after an hour of exposure.

Fig. 6 shows the yield of covalent complex formed as a function of time, when [14C]Phe-[~4U]tRNAPhe was irradiated in the presence of phenylalanyl-tRNA synthetase. The incorporation reaches a plateau when approximately 15 of the modified tRNA is bound to the enzyme. It should be emphasized that a con- comitant loss of the acylation activity of phenylalanyl- tRNA synthetase is observed, equivalent to the amount of bound tRNA and that incomplete joining is ob- served. Inactivation of the enzyme cannot be im- plicated but more likely a photoinactivation of the thiol groups occurs before the cross-linking. Indeed, addition of a fresh portion of [s4U]tRNAPhe results in further joining (up to 50 %) of the residual and un- crosslinked tRNA. Again, inactivation of the acylation activity of the enzyme somewhat parallels covalent bond formation.

On the other hand, during the whole course of the experiments no loss of the ATP-PPi exchange activity is observed, indicating that crosslinking of the nucleic acid molecule to the protein leaves the catalytic centre intact.

The joining of [s4U]tRNAPhe to the enzyme is really specific for the tRNA binding site, since addition of a 15-fold excess of native tRNAPhe almost completely inhibits the cross-linking reaction (residual incorpora- tion 0.52 %). The modified tRNAPhe used in this study contained a rather large number of s4U residues, in order to cover a maximum number of interaction sites. These residues were statistically distributed along the tRNA molecule, as was shown by the analysis of elution pattern of a TI ribonuclease hydrolysate on a DEAE-cellulose column : five major thiouridine peaks were found, out of nine possible fragments.

After cross-linking and pancreatic ribonuclease hydrolysis of the covalent complex, the bond oligo-

487

I

Fig. 6. Cro.wlinkmng of phenyiulunyl-tRNA syniiietasr to [ ' 'CJPI~~,- [s4U]tRNAphe at 330nm versus time of irradiation. (U) Enzyme inactivation; (0) cross-linking. At the point indicated by the arrow fresh tRNA was added

nucleotides were post-labelled as described above. The efficiency of labelling was only 2.3% but was assumed to be statistical. Fig. 3 shows the analysis of the modified enzyme by sodium dodecylsulphate/ polyacrylamide gel electrophoresis: up to 95 % of the radioactivity is located at the level of the p subunit.

Covalent Binding of tRMA:? to Phenylulunyl-tRNA Synthetuse by Schiff s Base Formation

Fig. 7 shows the extent of covalent binding after stabilization of the Schi fs base by borohydride reduc- tion. A very efficient incorporation is observed, since around 0.7 mol tRNA is bound/mol enzyme (35% yield with respect to the available binding sites). As can be seen, this covalent labelling appears to be absolutely specific for the tRNA binding site, since the addition of a tenfold excess of native tRNAPhe almost completely inhibits the labelling reaction. It must be emphasized that the inhibition of the acylation activity of phenylalanyl-tRNA synthetase strictly fol- lows the formation of the covalent bond, as shown in Fig. 7, whilst the ATP-PPi exchange activity remains unchanged. Since the formation of a Schiffs base is a reversible reaction, it had to be checked whether the covalent incorporation of tRNAPhe could be reversed. A control experiment was run in which, after 150 min of incubation, an aliquot was withdrawn, diluted tenfold in the presence of a 50-fold excess of native tRNAPhe and filtered on a nitrocellulose mem- brane, without prior reduction by borohydride. As shown in Fig.7, this results in a partial loss of the labelling by the tRNA, together with a regain of acylation activity of the enzyme. The fact that the labelling was not completely reversed can be explained by the slow rate of Schiffs base formation and re-

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488 Yeast Phenylalanyl-tRNA Synthetase

40 r I

._ I j 8 % . - 0

- 20 n c 3 0 D

g, 10 z o 100

A I ._ .- I

U 80

4 M)O 30 60 90 I20 150 180

Time (rnin)

Fig. 7. Covalent incorporation of' oxidized tRNAPh' in phenylulmyl- tRA'A synthetase by Schijf s base formution. (A) Extent of covalent binding of oxidized [3H]tRNAPhe to phenylalanyl-tRNA synthetase in the absence (e-e) or in the presence (t-.) of a tenfold excess of native tRNAPh'. 2.7 pM enzyme, 5.4 pM oxidized [3H]- tRNAPhe (120 counts x min-' x pmol-'). The Schiffs base formed during incubation was stabilized by borohydride reduction prior to adsorption on nitrocellulose membranes. After 150 min of in- cubation, the reversibility of the addition reaction was tested (0) by dilution of an aliquot in presence of a 50-fold excess of native tRNAPhc and filtration on nitrocellulose membrane omitting the borohydride reduction. The arrows shows the resulting decrease of covalent binding. (B) Variation of enzymatic activities upon Schiffs base formation. (O--O) Acylation activity of the enzyme; (D-0) ATP-PPi exchange activity. As above, the arrow shows the activity regain upon reversion of SchiFs base adduct

version. All these results are thus perfectly in agree- ment with the postulated mechanism of covalent incorporation by Schiff s base formation and suggest the existence of a lysyl group in the close vicinity of the active site, which is able to react with the 3'-ter- mind adenosine of tRNAPhe after periodate oxidation. The fact that the covalent labelling cannot be made to reach completion (only 35% of the available sites reacted after 150 min) can be understood, since the formation a Schiffs base is equilibrated, the water elimination between both reactants being antagonized by the solvent. As borohydride not only stabilizes the Schiffs base but also reduces free aldehydic groups, the reduction will freeze the equilibrium instead of displacing it towards the covalent adduct. The use of a milder reducing agent like cyanoborohydride, which is known under given conditions to reduce imino bonds without affecting aldehyde groups, should allow an efficient stabilization of the Schiffs base together with a displacement of the equilibrium in favour of the covalent adduct 1191. Preliminary experiments

have been performed in order to improve the yield of covalent labelling by tRNAEr upon reduction with cyanoborohydride. Up to now, no significant im- provement has been obtained, but this may result from non-optimal reaction conditions, since in the case of E. coli methionyl-tRNA synthetase, the use of cyanoborohydride allowed a 100 labelling effi- ciency (S. Blanquet, personal communication).

In order to identify the subunit(s) responsible for the tRNA binding, the covalent complex recov- ered by Sephadex filtration was analysed by sodium dodecylsulphate/polyacrylamide gel electrophoresis after pancreatic ribonuclease hydrolysis of the bound tRNA. Fig. 8 shows that, here also, only the a subunit ( M , 63000) of the enzyme exhibits a significant label from tritiated 3'-terminal adenosine.

All the above experiments show that tRNAPhe appears to interact exclusively with the 1 subunit of the enzyme, whatever the selectivity of the cross-linking procedure used : either nonspecific ultraviolet irradia- tion, irradiation of 4-thiouridine residues statistically introduced in the tRNAPhe molecule, or specific methods like irradiation in the wye base absorption band and Schiffs base formation with the oxidized 3'-terniinal adenosine. They are also in perfect agree- ment with previous results of Fasiolo et al. [2] who showed that selective proteolysis of the 1 subunit resulted in the complete loss of the tRNA binding ability and that, conversely, tRNA strongly protected the B subunit against specific proteolysis,

Reaction of Pheizylulanyl-tRNA Synrhetuse with Pyridoxal 5'- Phosphate

Pyridoxal 5'-phosphate has been successfully em- ployed as a reagent for identification of lysyl groups at the active centre for several enzymes [20,21]. Re- cently, inhibition of isoleucyl-tRNA synthetase has been described [21] which does not obey a classical active-site-directed irreversible inhibition. Fig. 9 shows the effect of this compound on the activities of phenyl- alanyl-tRNA synthetase. While the aminoacylation reaction is severely inhibited by 1 mM reagent, ATP-PP; exchange remains unchanged. Complete irreversible inactivation was obtained at 5 mM pyridoxal 5'-phosphate, followed by reduction with borohydride. The addition of the small ligands, either individually or together to form the adenylate in situ, at concentrations which saturate the enzyme does not afford any protection, while tRNAPhe completely protects the aminoacylation activity.

In an attempt to determine the number of residues responsible for the complete loss of the aminoacylation activity, the enzyme (20 pM) was modified by the reagent, reduced with sodium borohydride and dial- ysed exhaustively. In addition to the maximum ab- sorbance at 280nm, the modified enzyme had an

Page 9: Yeast Phenylalanyl-tRNA Synthetase : Affinity and Photoaffinity Labelling of the Stereospecific Binding Sites

M. Baltzinger, F. Fasiolo, and P. Remy 489

0 1 2 3 4 5 6 7 8 - (+ I Distance of migration (cm)

Fig. 8. 1il~'iitif;;c.NtiolIitioii of t h ~ subunit ofphenylulan~l-tRhlA synthrtuse involved in Schif".s base ,formcition initlz oxidized J3H]tRhrA'"' by sodiuni dodecj./su~liatr/}~olyucr~Ilanzide gef electrophoresis, uytcr pancreatic rihonuc~lerisr Ii.yrirofysis o/ tlir covulent complex

2 4 6 8 10 12 14 18 Time (min)

Fig. 9. Tii7w wur.sv of iiioc~rilation of'phenylufan~I-tRNA synrl7etuse by 1 mM pyridox-ul S'-phospIiute, in the presence of 10 mM MgClz at 25 ,C. (B) ATP-PP, exchange activity, no added substrate; (0) aminoacylation activity, no added substrate; (0) with 1 mM phenylalanine; (0) with 5 mM ATP; (A) with 1 mM phenylalanine and 1 mM ATP; @ with 10 p M tRNAPhe-C-C-A; (A) with 10 p M tKNAPhe-C-C

additional peak at 325 nm, suggesting that the modified amino acid is a lysyl residue [21]. Using a molar ab- sorption coefficient of 1.015 x lo4 M- ' cm-' for the modified lysyl residue [21] a stoichiometry of 22 mol lysine residues/mol enzyme could be calculated. By this spectrophotometric technique no particularly reactive residue could be detected. We therefore turned to another reagent, the 2',3'-dialdehyde derivative of

I I

1 2 Inhibitor incorporated (mol imol enzyme)

Fig. 10. Sroichiometry u/' irreversible inactivutiorz n f ph~~n~ lu lu r iy l - rRh'A synthetuse by Mg-oATPZ- at 25 C. The enzyme (4.9 pM) was incubated with 0.53 mM of inhibitor (A) or with 0.266 mM inhibitor (B). The open (0, U) and full (0, m) symbols correspond to two different sets of experiments. A protection experiment was performed in the presence of 10 pM tRNAPhe (A) in B

ATP (oATP), to solve this problem. This compound, which was reported to be an effective affinity label in the case of several aminoacyl-tRNA synthetases from E. coli [22], was prepared as described in [23]. This reagent also exhibits a different effect on the activities of phenylalanyl-tRNA synthetase. At 4 mM oATP, the aminoacylation activity is completely abolished after 1 h of incubation while 70% residual ATP-PPi exchange activity is present. With the use of ['4C]oATP we tried to modify selectively the lysine groups in- volved in the aminoacylation activity. Fig. 10 shows a plot of the loss of activity versus the number of oATP residues incorporated per mole of enzyme at two concentrations of inhibitor. Several conclusions can be drawn, as follows.

(a) Since the ATP-PPi exchange activity remains unaffected, it may be deduced that the observed labelling by oATP does not occur at the ATP binding subsite.

(b) The inhibition of the acylation activity of the enzyme suggests that oATP simply reacts with some lysine group essential for the acylation of the tRNA. The important amino acid residue is probably located close to the attachment site of the 3'-terminal adeno- sine, since while native tRNA completely protects this

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490 Yeast Phenylalanyl-tRNA Synthetase

lysyl group, tRNAPhe-C-C only partially prevents the modification (Fig. 9). These results are perfectly con- sistent with the specific modification of phenylalanyl- tRNA synthetase by tRNA;: upon Schiff s base formation.

(c) As can be seen from Fig.10, a maximum of two residues per mole of tetrameric enzyme are in- volved in the acylation activity. However, working with a lower concentration of oATP (0.266 mM), it can be seen that the first reacting residue is not in- volved in the acylation activity, since up to 0.7 residue can be modified without any loss of activity. The first molecule of oATP incorporated thus appears to react rapidly with the enzyme outside the active site, this modification leaving the acylation activity intact. The chemical modification at the level of the active site only takes place in a second step and the striking feature is that a stoichiometric inactivation is observed when 2mol oATP have reacted with the enzyme. Since the binding of the first one does not inactivate the enzyme, the complete inactivation has to occur upon the modification of a single site per c 4 2 mole- cule. This result is somewhat surprising since it is known that phenylalanyl-tRNA synthetase is a func- tional dimer and, at least in the absence of other ligands, binds 2 mol tRNA with the same affinity [l]. Since we previously reported an asymmetric behaviour of the enzyme during catalysis [24], we are therefore led to the conclusion that either ATP alone (or its analogue) may induce the observed asymmetry or this asymmetry already exists in the binding of tRNA, although the affinities cannot be distinguished.

Labelling of Plzenylalanyl-tRNA Synthetase by Phenylalanine upon Irradiation at 248 nm

Covalent incorporation of phenylalanine in phenyl- alanyl-t RNA synthetase was studied by irradiation at 248nm of complexes between the enzyme and free amino acid or aminoacyl adenylate or aminoacyl- tRNA. The results are summarized in Table 3. A salient feature is the low yield of incorporation ob- served, except at high concentration of free ligand. The covalent labelling was found to be equally dis- tributed among CL and f l subunits in the case of enzyme- amino-acid and enzyme-adenylate complexes. This raises the question as to whether or not the labelling reaction is specific. Indeed, as shown on Fig. 11, the inactivation of phenylalanyl-tRNA synthetase is much more important than phenylalanine incorporation, which leaves the possibility for the excited amino acid to be covalently bound even to inactive enzyme on a fortuitous encounter of both molecules. If the labelling by phenylalanine was restricted to the binding site specific for this amino acid, one would expect an in- crease of the labelling yield with the amino acid con- centration up to a point where all binding sites are

Table 3. Yield oJ cross-link between pl~enyluluny/-tRNA synthetase andphenylufunine and distribution of the labelling on the constitutive subunits of the enzyme In expt 1, free ['H]phenylalanine was used in the absence or presence of acylated tRNAPh'; in expt 2, [3H]phenylalanine bound to tRNAPh' was used in the absence or in the presence of free competitive phenylalanine: in expt 3, [3H]phenylalanine joined to the adenylate was used. The enzyme concentration in all experiments was 1.2 pM, that of Phe-tRNAPh' (when present) was 2.4 pM

Expt State of Concn Phe- Phenyl- Labelling [3H]phenyl- of free tRNAPhe alanine of subunit a 1 an i n e phenyl- present bound

alanine E B

mM mo1/100 sites

1. Free 0.05 - 1.2 56 44 5.0 - 22 47 53 0.05 + 1.26 51 49 0.5 + 7.14 51 49 5.0 + 45.2 41 53

2. Bound to 0 + 0.23 16 84 tRNA"he 0.05 + 0.19 19 81

0.5 + 0.12 18 82 5.0 + 0.22 9 91

3. Bound to AMP 0 - 0.4 35 65

Fig. 11. Covdent incorporation of phenylalanine into phenykdanyl- tRNA synthetase upon irradiation at 248 nm. 1.2 pM enzyme, 50 p M [3H]phenylalanine(785 counts x min-' x pmol- '). (0-0) Extent of covalent binding; (-0) inhibition of the acylation activity of the enzyme

saturated. Table 3 shows that this prediction is not fulfilled since increasing the amino acid concentration from 0.5 mM to 5 mM results in a sharp increase of the labelling efficiency. Now, a 0.5 mM concentration should be sufficient to saturate the normal binding sites for phenylalanine ( K d < 100 pM). This result could thus be interpreted as a lack of specificity of the covalent incorporation ; but it can also be understood

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M. Baltzinger, F. Fasiolo, and P. Remy 49 3

if the excited state of phenylalanine is sufficiently long- lived that a molecule of amino acid excited in the free state in solution may have the opportunity to be exchanged with another molecule of amino acid, bound to the enzyme, before coming back to the ground state or reacting with the solvent. In this case, the proba- bility of covalent bond formation at the acceptor site should be proportional to the total concentration of excited molecules and not only to the fraction of excited amino acid present at the enzyme active site. Another possibility would be the existence on phenylalanyl- tRNA synthetase of another set of binding sites for phenylalanine with a lower binding constant. Pre- vious results of Raffin and Remy [25] have already suggested this possibility.

It has to be remarked that upon irradiation of the aminoac yladenylate-enzyme complex, an almost equiv- alent distribution of the amino acid among a and p subunits was observed. Now, under these conditions, the specificity of the labelling reactions should be much higher, since the adenylate has a high affinity for the enzyme and if some hydrolysis of the adenylate occurs, the liberated phenylalanine will be present in the mixture in low concentration, which should preclude any non-specific labelling upon bimolecular reaction. We thus believe that the observed distribution of the phenylalanine residue on a and p subunits reflects equivalent probabilities of reaction between excited phenylalanine and amino acid residues be- longing either to a or f i subunit(s). Previous results of Raffin and Remy [25] had shown that upon photo- oxidation of phenylalanyl-tRNA synthetase in the presence of rose Bengal, phenylalanine was only significantly incorporated into the cx subunit. The equal probabilities of the activated amino acid reacting with either a or p subunit might just result from a spatial vicinity of amino acids from both chains at the level of the active site. Previous results of Murayama et al. [17] already suggested that the active centre of phenylalanyl-tRNA synthetase might be close to a contact area between a and p subunits, since the thiol groups essential for the acylation activity of the enzyme could be involved in the formation of a di- sulphide bridge between a and p subunits.

A striking difference is observed when phenyl- alanine is bound to the tRNA prior to incorporation into phenylalanyl-tRNA synthetase. As shown on Table 3, the labelling almost exclusively occurs on the /? subunit. This result is not surprising, since it was shown above that the 3'-terminal adenosine of tRNAPhe interacts with this subunit. The binding site on the /? subunit of the phenylalanine residue linked to the tRNA is clearly distinct from that of the free amino acid since, as shown in Table 3, no competition can be observed between free amino acid and aminoacyl- tRNA in either type of crosslinking experiment. These results support the hypothesis that the amino acid,

when linked to the tRNA, is no longer present in its normal stereospecific binding site, as already suggested by Yarus [26] and Bonnet 1271. Furthermore, the in- cubation of phenylalanyl-tRNA synthetase with hr- bromoacetyl-phenylalanyl-tRNAphe results in the crosslinking of the amino acid residue almost ex- clusively to the /? subunit (Fasiolo, unpublished results). As any modification of the NH2 group of the amino acid precludes the binding of the modified amino acid in its normal accepting site, the covalent complex formed has to occur in a second site distinct from the amino-acid-activation site.

Photosensitized Cross-linking of ATP to Phenylalanyl-tRNA Synthetase

No incorporation of ATP into phenylalanyl-tRNA synthetase could be observed upon monochromatic ultraviolet irradiation, at 240, 248, 265 or 280 nm, although a sharp inactivation of the enzyme occurred during the irradiation. The use of a germicide lamp to irradiate ATP-enzyme complexes, according to Yue and Schimmel [28], resulted in our hands in an efficient joining of ATP to phenylalanyl-tRNA syn- thetase but also to bovine serum albumin, suggesting a lack of specificity. A potent photoaffinity marker, adenosine 5'-[y-(p-azidoanilido)]triphosphate has been synthesized by Budker et al. [29] and used by Akhver- dyan et al. [30] to label tryptophanyl-tRNA synthe- tase. But in the case of E. coli phenylalanyl-tRNA synthetase, Ankilova et al. [31] showed that the ob- served labelling was not specific. We thus turned to the acetone photosensitized crosslinking technique described by Sperling et al. [32 - 341. The irradiations of enzyme/ATP mixtures were performed in the pres- ence of 5 % acetone, at 313 nm, a wavelength which offers a good compromise between cross-linking effi- ciency and enzyme inactivation. As shown in Fig. 12, the irradiation outside the absorption band of acetone (335 nm) does not result in any labelling or inactivation of the enzyme. Upon irradiation at 313 nm, an efficient covalent incorporation of ATP occurs (45 % yield, mol/mol; 22.5 % mol/site after 2 h), but the rate of inactivation of the ATP-PPi exchange activity is higher than the rate of labelling. Thus, the specificity of the joining had to be checked. This was done using a mixture of phenylalanyl-tRNA synthetase and [y-32P]ATP irradiated either in the absence or in the presence of 1 mM phenylalanine. In the presence of the amino acid, the formation of the adenylate (10 units pyrophosphatase/ml were added to ensure a complete reaction) results in the loss of the pyro- phosphate containing the labelled y-phosphate. Under these conditions, the active site is blocked by an adenylate molecule which does not contain any radio- activity since the j3 and y phosphate of ATP have been eliminated. But the excess of ATP is still intact,

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492 Yeast Phenylalanyl-tRNA Synthetase

" 0 60 120 0 60 120 0 60 120

nrne (min)

Fig. 12. Photo.sensitized incorporation id ATP into phcwylulunyl-tRhJA synt1wtu.w upon irrudiation in the presence of 5 % acetone at several wuvc,frngths uhove 300 nm. 37 pM enzyme, 500 pM [I4C]ATP (165 counts x inin-' x pmol-I). (0-e) Extent of covalent binding; (0--0) inhibition of ATP-PPi exchange activity of the enzyme

since the formation of adenylate is stoichiometric to the amount of enzyme present. Any non-specific cross- linking reaction can thus proceed, as in the absence of phenylalanine and the amount of radioactivity incorporated in presence of phenylalanine will thus be a direct measure of the lack of specificity of the photosensitized labelling. As shown in Fig. 13, this non-specific covalent binding appears negligible since only 7 % (mol/moI) of cross-linking occurs in the presence of amino acid. Furthermore, we controlled that the presence of phenylalanine did not prevent the incorporation of ATP by any side effect (for in- stance by deactivation of the triplet excited state of acetone), precluding the covalent binding of ATP, since [U-14CJATP was incorporated with the same efficiency in the absence or in the presence of phenyl- alanine (Table 4). Thus the covalent labelling ob- tained appears to be specific for the ATP binding site.

After separation of the covalent complex from the excess of ATP by molecular sieving on Sephadex G-50, the subunit responsible for the covalent binding of ATP was identified by sodium dodecylsulphate/poly- acrylamide gel electrophoresis of the labelled enzyme. AS shown in Fig. 14, only the I( subunit exhibits a significant labelling. After a long time of irradiation, labelled polymers could be detected at the top of the gel, probably resulting from intermolecular cross- linking of the protein.

CONCLUSION

The experiments reported above show that among the ligands of yeast phenylalanyl-tRNA synthetase, the tRNA and ATP exclusively interact with the

0 10 20 30 40 50 Time (min)

Fig. 13. Control of tlie sprcifkity 01 rRe plrorosensiti~c~d covulent in- corporution oJ[y-"P]ATP upon irradiation at 313 nm in thi~prescvic~c~ o / 5 % uceton~. 9.6 pM enzyme, 500 pM ly-32P]ATP (400 counts x min-' x pmol-I). (O--O) Incorporation in the absence of phenylalanine; (0- -0) incorporation in the presence of 1 mM phenylalanine

Table 4. Control o/photo.sansit~~ed incorporation of A TP into i~henyl- ulunyl-rRNA syntiietasc~ upon ir-radiation ut 313 nm in the ptywi1c.c of'5'7~ acetone

Concn of [14CJAMP Yield of bound covalent

enzyme [14C]ATP Phe complex - -- ~ - -

PM

5 1 53 9 6 500 -

9 6 500 1000 4 75 50

_ _ ~

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M. Baltzinger, F. Fasiolo, and P. Remy

A

493

B

10 ‘20 30 60 K) 20 30 60 Time of irradiation (rnin)

Fig. 14. Identification by sodium dodrcylsu~phate~polyacrylamide gel electrophoresis c$ the subunit of phenylalan$-tRhrA synthrtase involved in tltr photosensitized covalent binding of ATP upon irradiation at 313 nm. (A) Staining of the electrophoregram by Coomassie blue; (B) autoradiography of the electrophoregram

smaller subunit of the enzyme (p subunit). The amino acid was also found cross-linked to that subunit, when bound to the tRNA. But when the phenylalanine was free or engaged in the adenylate, an almost sym- metrical distribution of the ligand on both subunits was observed. As already discussed, this could result from some lack of specificity in the binding reaction. We do not favour this hypothesis, since the important enhancement of the affinity for the phenylalanine when engaged in the adenylate does not modify significantly the labelling pattern. This symmetrical binding of the amino acid to c: and ,8 subunits could thus reflect a spatial vicinity of both types of chains at the level of the amino acid binding site. As already discussed, this hypothesis is supported by the pos- sibility of formation of a disulphide bridge between 0: and f l subunits, involving the essential thiol groups of phenylalanyl-tRNA synthetase. Whether or not this spatial vicinity of both types of subunits at the level of the active site is important for the activity is not clear yet. But it must be emphasized that at least one example is known where an active site is composed of two different polypeptide chains [35].

Our results also show the importance of lysine residues for the acylation activity of the enzyme. These lysine groups most probably lie close to the binding site for the 3’-terminal adenosine of the tRNA. It must be emphasized that the lysyl groups involved in the reaction with tRNA,,, oATP and pyridoxal phosphate are different from the lysyl groups which have been shown to be present at the activation site of bacterial aminoacyl-tRNA synthetases by Fayat et al. [22]. In- deed, as mentioned above, the ATP-PPi exchange activity remains unaffected, while the acylation activity is strongly inhibited. Obviously, our results do not

allow a conclusion on whether the important lysyl groups really play a role in the catalytic mechanism, or whether they are simply involved in allowing a better fit of both molecules in the region of the active site (e.g. by exchanging an ionic bond with a phosphate group of the tRNA). The presence of such a lysyl residue at the vicinity of the acceptor end of tRNA might be an important common property of amino- acyl-tRNA synthetases, since it has already been shown that E. coli isoleucyl-tRNA synthetase was inhibited by pyridoxal phosphate.

We thank Dr Lapidot (Jerusalem) for providing us with [s4U]tRNAPh‘. The authors also wish to acknowledge the technical assistance of N. Menard and G. Nussbaum and the help of M. Schlegel in purifying yeast IRNA’”‘. This work was partly sup- ported by grants from the DPligagation GPnerale u Ju Recherche Scienrifiyue et Technique and from the Commissariat ri I’Energie Atomique. We thank Professor J . P. Ebel for continuous interest in this work and Professor Y. Boulanger for help in preparing the manuscript.

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494 M. Baltzinger, F. Fasiolo, and P. Remy: Yeast Phenylalanyl-tRNA Synthetase

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M. Baltzinger, F. Fasioio, and P. Remy*, Institut de Biologie Molkculaire et Cellulaire du C.N.R.S., 15 Rue Rene Descartes, Esplanade, F-67084 Strasbourg-Cedex, France

* TO whom correspondence should be addressed.

Note Added in Proof Specific labelling of phenylalanyl-tRNA synthetase by tRNA:’ through Schiffs base formation could recently be made to reach completion upon incubation of the enzyme with the modified tRNA under the following conditions: 30 mM veronal buffer pH 8.5, 3.2 pM phenylalanyl-tRNA synthe- tase, 7 pM tRNA::, 5 mM cyanoborohydride. The incubation was carried out at 37 “C. After 3 h of incubation, the yield of covalent complex was higher than 70 % (mol/site) corresponding to 1.4- 1.5 mol of tRNA bound per mol of enzyme. The observed inhi- bition of the acylation activity of the enzyme was strictly stoichio- metric to the covalent binding. The more alkaline pH of the incu- bation mixture favours Schiff s base formation by increasing the fraction of lysyl group in the - NH2 form and increases the selec- tivity of imino-bond reduction by cyanoborohydride.

The possibility of covalent complex formation between amino- acyl-tRNA synthetases and oxidized cognate tRNAs seems to be general, since it was also observed in the yeast valyl and aspartyl systems (R. Giege, personal communication).