- trna complexes in several other systems : methionyl-trna

Transcription

1 Eur. J. Biohem. 24 (1972) The Mehanism of Reation of Methionyl-tRNA Synthetase from Esherihia oli Interation of the Enzyme with Ligands of the Amino-Aid-Ativation Reation Sylvain BLANQUET, Guy FAYAT, Jean-Pierre WALLER, and Motohiro IWATSTJBO Laboratoire d'enzymologie, and Laboratoire de Gbnbtique Molbulaire, Centre National de la, Reherhe Sientifique, Gif-sur-Yvette (Reeived September 29,1971) The interations of methionyl-trna synthetase, a tetrameri enzyme, with various ligands involved in the methionine-ativation reation (methionine and analogues, ATP, AMP, PPi, methioninyl adenylate and methionyl adenylate) were examined by equilibrium dialysis, absorbane differene spetrosopy and fluoresene measurements at equilibrium and in a stoppedflow apparatus. Two binding sites for methionine and four for ATP were found by equilibrium dialysis. The number of binding sites and the assoiation onstants of these ligands for the enzyme were not affeted by the presene of Mg2+ or EDTA. From ligand-indued %uoresene and absorbane variations, two binding sites were measured for methioninyl adenylate, a strutural analogue of methionyl adenylate. The interation of methionyl-trna synthetase with most of the ligands investigated gave rise to harateristi fluoresene variations of large amplitude. Thus, saturating onentrations of methioninyl adenylate and methionine enhaned the fluoresene emission by 40 /, and 260/,, respetively, while interation with hemially synthesized methionyl adenylate resulted in 270/, quenhing. ATP, AMP or PPi, alone or in ombinations, did not affet the emission spetrum. However, when ATP was added to enzyme saturated with methionine, the fluoresene level shifted from the initial value of 126O/, to 8Z0/,. Similary, additon of PPi to enzyme saturated with ATP and methionine raised the level of fluoresene from 82O/, to 1120/,. In none of the ases investigated did addition of Mg2+ or EDTA affet the final level of ligand-indued fluoresene modifiations. Titration experiments were onduted to measure the assoiation onstants of the ligands for the enzyme. The rates of formation of methionyl adenylate and of reversal of this reation by PPi were determined by stopped- flow measurements. Comparison of reation rates in the presene of Mg2+ or EDTA reveals a dramati enhanement of the rates of both reations in the presene of Mg%+. The speifi attahment of eah amino aid to its ognate trna(s) is a ruial, speihity-determining step in protein biosynthesis. The sequene of reations whih lead to formation of an aminoayl trna are atalyzed by the same enzyme, an aminoayltrna synthetase, apable of reognizing speifially both a given amino aid and its trna adaptor. The eluidation of the mehanisms whih underlie the speifiity and the atalyti funtions of aminoayltrna synthetases is the objet of many urrent investigations, using a variety of methods (for reent survey see [l]). Of the various tehniques available for determining the harateristis of the interation between aminoayl-trna synthetases and their ligands, that Enzyme. Methionyl-tRNA synthetase (EC ). whih involves equilibrium and kineti measurements of ligand-indued modifiations of enzyme fluoresene offers partiular advantages, provided that binding of ligands auses measurable hanges in the fluoresene parameters of the enzyme. First applied to the study of the trna-binding properties of valyl-trna synthetase by HQlbne et al. [Z], the method has sine been used to investigate enzyme - trna omplexes in several other systems : methionyl-trna synthetase (dimeri form) [3], seryltrna synthetase [4], phenylalanyl-trna synthetase [5] and arginyl-trna synthetase [6]. More reently, two reports on the appliation of the method to the study of the interation of aminoayl-trna synthetases with ligands involved in the amino aid ativation reation have appeared [7,8]. The fluoresene properties of the two enzymes investigated

2 462 Binding Properties of Methionyl-tRNA Synthetase Eur. J. Biohem. differed in their response to enzyme-ligand interations. While minor yet measurable fluoresene quenhing resulted upon interation of valyl-trna synthetase with its ligands [7], no detetable hanges in the fluoresene emission spetrum ourred in the ase of isoleuyl-trna synthetase [8]. In the latter ase, the binding properties of the enzyme were dedued indiretly, by measuring the effet of ligands on the rate of deay of the intrinsi fluoresene of the protein in the presene of 2.5 M urea. In the present work we have investigated the mehanism of the ativation of methionine by methionyl-trna synthetase, a tetrameri enzyme having apparently idential subunits of moleular weight [9,10]. The number of binding sites and the assoiation onstants for methionine and ATP were measured by equilibrium dialysis, and fluoresene tehniques were applied to detet formation of binary omplexes and to follow interations in the presene of more than one type of ligand. Stoppedflow measurements were performed to evaluate the kineti parameters of these interations, in the absene and in the presene of magnesium. The results reported learly indiate that fluoresene tehniques provide a valuable tool for haraterizing the various steps involved in the ativation of methionine by methionyl-trna synthetase. Formulation of a plausible reation mehanism will be defered until the possible influene of methionine-speifi trna on the ativation reation has been examined. MATERIALS AND METHODS Native methionyl-trna synthetase was purified from Esherihia oli K 12 strain EM arrying the F-32 episome as desribed earlier [Ill, and was stored in the presene of 50 /, glyerol at - 15 "C. Before eah series of experiments, glyerol was removed by gel filtration through Sephadex G-25 equilibrated with the appropriate buffer ontaining 10 mm 2-meraptoethanol. Protein onentration was determined from absorbane at 280nm, using the absorption oeffiient E: Zlm, = This value, whih was derived independently from the amino aid omposition of the protein, and from its refrative index relative to bovine serum albumin as standard [12], has sine been onfirmed by analysis of the interferene patterns obtained during ultraentrifugation of the enzyme, relative to ovalbumin as the standard [13]. When needed, solutions were onentrated by ultrafiltration in a Seletron Ultra- Thimble Apparatus (Shleiher & Shiill, Dassel). The ativities of the enzyme preparations used were in the range of units/mg in the ATP-PP, exhange assay, and 15 units/mg for the aminoaylation of trna. Assay onditions and definition of ativity units are as desribed earlier [Ill. All solutions ontaining methionine or its analogues and derivatives were supplemented with 10 mm 2-meraptoethanol to suppress formation of the orresponding sulfoxides. L-Methioninol and L-methioninyl adenylate [14] were kindly provided by Dr. R. Boissonnas. L-Methionyl adenylate was synthesized aording to Berg [15] and stored at - I5 "C. Immediately before use, samples were freed from ontaminating methionine and AMP by preparative paper eletrophoresis on Whatmann 3-MM at 2 "C in 20 mm sodium itrate buffer at ph 3.1 (60 V/m for 90 min). Elution and appropriate dilutions were ahieved at 4 "C with water at ph 3.7 (HC1). ~-[Me-~~C]Methionine (50.5 mci/mmol) and [14C]- ATP (220 mci/mmol) were purhased from the Commissariat b 1'Energie Atomique (Frane). The former was freed from methionine sulfoxide by preparative thin-layer hromatography on ellulose, using the solvent system see-butanol- 3 ammonia (3:1, vlv). [I4C]ATP was purified by high-voltage eletrophoresis [IS], eluted with water and stored at - 15 "C. L-Methionine, D-methionine and ATP (sodium salt) were purhased from Mann Researh Laboratories, and 3-methylthiopropylamine from Eastman Kodak. Fresh solutions were prepared for eah series of experiments. One of the following buffers was used in the different experiments, as indiated in the orresponding legends to the figures: Buffer A: 20 mm potassium phosphate ph 7.6; Buffer B: 100 mm potassium phosphate ph 7.6, 5 mm MgCl,; Buffer C: 20 mm potassium phosphate ph 7.6, 100 mm KCl, 0.5 mm MgC1,; Buffer D: 100 mm potassium phosphate ph 7.6, 1 mm EDTA (Na); Buffer E: 20 mm potassium phosphate ph 6.0. All buffers ontained 10 mm 2-meraptoethanol. Equilibrium Dialysis Equilibrium dialyses were arried out at 4 "C in slowly rotating leuite ells [17] equipped with prewashed Visking dialysis membranes. The ell ompartment whih reeived the ligand solution had a apaity of 0.88ml and was filled to 0.4ml. Two types of enzyme ompartments were used, with apaities of 0.88 ml (type 1) or 0.15 ml (typeii) filled to 0.4 ml or ml, respetively. The time required for equilibration was 4 h for methionine and 14 h for ATP. Samples were removed after 12 and 24 h, respetively. Conentrations of ligands were hosen suh that the ratio of bound to free ligand was not less than 0.1 at the highest onentration of ligand tested. Solutions were prepared by mixing a onstant amount of the radioative ligand with inreasing amounts of the orresponding unlabelled one. No loss of enzymati ativity was enountered during dialyses.

3 Vo1.24, No.3,1972 S. BLANQUET, G. FAYAT, J.-P. WATLER, and M. IWATSUBO 463 Radioative samples were ounted in 20ml of Bray solution [18] in a Pakard Sintillation Counter. No signifiant quenhing of radioativity was observed under the experimental onditions used. Absorbane- Di f f erene Spetro photometr y Differene spetra were reorded at 24 "C in a Cary-I5 Spetrophotometer using two-ompartment quartz ells (Hellma) with a light path of m. The referene and sample ells ontained 0.8ml of the same protein solution (absorbane lose to 2 at 280 nm). The base line (protein versus protein) and the spetra were traed on the expanded sale. Aliquots of 5pl of a solution of L-methionhyl adenylate in buffer were then added to the sample ell and 5p1 of buffer to the referene. AA was measured as the maximal effet between the extremes after eah addition of ligand. Fluoresene Measurements Fluoresene measurements at equilibrium were performed at 24 "C in a sensitive spetrophotofluorimeter [19] equipped with a stabilized 450-watt xenon lamp (Osram), two monohromators (Baush and Lomb), an EMI-9558 &A photomultiplier and an x-y hart reorder. The fluoresene was exited at 295nm and the emission reorded at 328nm. The quartz ell (1 x 0.4 m, Hellma) was filled with 0.8 ml of the enzyme solution. Saturation urves were obtained by adding aliquots of 5 pl or 10 pl of the buffer ontaining the ligand under study. The titration urves, orreted for dilutions and for the sreen effet when neessary, show the maximal intensity of the fluoresene of the enzyme in the presene of inreasing onentrations of ligands. Stopped-flow measurements were arried out at 17 "C in a Durrum-Gibson apparatus equipped with a Tektronix-564 storage osiuograph and a 3A7 differential omparator. For eah traing, 0.2 ml of a solution of enzyme (0.44pM) in the appropriate buffer was mixed with an equal volume of the same buffer ontaining the ligand under study. All onentrations given in the text orrespond to final values after mixing. The dead time of the apparatus was 6 mse. Appropriate wavelengths were seleted using a stabilized xenon lamp (450 watt, Osram) as a light soure with a Jobin-Yvon grating monohromator (HRSB). The protein fluoresene was exited at 295 nm and observed at 90 " of the inident beam through a ut-off filter, as measured by the photomultiplier (EM19558 &A) at appropriate sensitivities. A variable apaitor was added in order to redue the photomultiplier noise. The rate onstant z-1 for eah binding reation was evaluated from enlarged photographs of the displays on the sreen of the storage osillograph, by measuring the slope of first order plots. In the ase of the bimoleular reations, the pseudo fistorder reation onditions used allow the determination of the kineti onstants aording to the following relations : E'E.S E + S T when the onentration of ligand, S, is in large exess over enzyme onentration. If [ESIeq is the onentration of the omplex at equilibrium and [ES] the onentration at time t, then Atomi-Absorption Spetrosopy Magnesium onentrations was determined using a Perkin-Elmer 290-B spetrophotometer. The instrument was alibrated with a referene solution ontaining 0.1 pg/ml of MgC1, in water. A solution of the enzyme in buffer A was dialyzed for 18 h at 4 "C against 2000 volumes of buffer A, and then diluted with the external dialysis buffer to final onentrations of 6.70, 3.34, 2.68 and 1.67 absorbane units/ml at 280 nm. The magnesium onentration of buffer A and of eah of these solutions was determined using the referene value. RESULTS EQUILIBRIUM MEASUREMENTS Equilibrium Dialysis Binding of L-Methionine and; of ATP to the Enzyme. The interation of L-methionine and ATP with methionyl-trna synthetase was measured separately by equilibrium dialysis. The number of binding sites and the assoiation onstants for these two ligands, dedued from Sathard plots (Fig. 1 and 2) of measurements performed under Werent experimental onditions, are summarized in Table 1. The results indiate that the tetrameri enzyme (moleular weight ) an bind two moleules of methionhe or four of ATP. No signifiant differenes in the number of binding sites and in the assoiation onstant for methionine and ATP were found when measurements were performed in the presene of Mg2+ or EDTA. The orresponding dissoiation onstants for methionine and ATP do not differ signifiantly from their Mihaelis onstants in the ATP-PPI exhange reation. Fluoresene and Absorbane Measurements Binding of L-Methioninyl Adenylate to the Enzyme. The bin- of methioninyl adenylate, a strutural

4 464 Binding Properties of Methionyl-tRNA Synthetase Eur. J. Biohem. Table 1. Number of binding sites and affinity onstants of mthionyl-trna synthetase for methionine and ATP, determined by equilibrium dialysis Experimental onditions are desribed in Methods Ligand L-Methionine L-Methionine ATP ATP Buffer A B C D Conn range Enzyme Type of Number of of ligand onn ell used binding sites lo-& x K. IrM IrN 12 I 12.8 I1 100 I1 85 I1 M-' Number of binding sites,n 2 Number of binding sites In - E L A I o 1.5 L - Methioninyl adenylate (nmol) Number of binding sites,n Bound methionine (FM) Fig.1. Sathard plot of results of the equilibrium dialysis of L-methionine and enzyme (12.8 pm) in buffer B (see Table 1) Number of binding sites,n L-Methioninyl adenylate (nmol) Fig. 3. Titration of methionyl-trna synthetase (0.5 nmol) with L-methioninyl adenylate in buffer A. Experimental onditions are desribed in Methods. (A) Maximum of the fluoresene emission spetrum as a funtion of ligand added. (B) Normalized fluoresene intensity as a funtion of ligand added Fig.2. Sathard plot of the results of the equilibrium dialysis of ATP and enzyme (0.1 mm) in buffer C (see Table 1) analogue of methionyl adenylate, markedly affets the fluoresene of the enzyme. The high affinity of this analogue for the enzyme, first demonstrated by kineti experiments [14], thus allows an aurate determination of the number of binding sites by following the inrease in fluoresene intensity as a funtion of ligand onentration. A saturating amount of the ligand auses a 4QQ1, inrease in protein fluoresene and a red shift of 7 nm in the emission spetrum (Fig. 3). Similar titration experiments were performed by Herene absorbane spetrosopy, based on the observation that the binding of methioninyl adenylate auses a measurable hange in the absorption spetrum of the enzyme (Fig.4 and 5). The number of binding sites for methioninyl adenylate dedued from both types of measurements is lose to two. Binding of L-Methionine to the Enzyme. Saturating amounts of methionine ause a 26OlO inrease in

5 Vo1.24, N0.3,1972 S. BLANQUET, G. FAYAT, J.-P. WALLER, and M. IWATSUBO ' Wavelength (nm) Fig. 4. Differene-absorption spetrum obtained upon addition of L-methioninyl adenylate to methionyl-trna synthetase. The referene ell ontained a solution of enzyme (8 a). The sample ell ontained the same solution of enzyme with a saturating amount of the ligand. The base line (protein solution in buffer A in both ells) was subtrated Fig. 7. Titration of methionyl-trna synthetase (0.2 pm) zvith ATP in the presene of a saturating ammount of L-methionine (0.6mM) in buffer A. The shape of the urve is not affeted by prior addition of AMP (0.5 mm) to the enzyme solution Number of binding sites,n ZI._ - v) " P N i P L - Methioninyl adenylate (pm) Fig. 5. Titration of methionyl-trna synthetase with L-methwninyl adenylate measured by dif ferene-absorbane spetrosopy. For onditions, see Fig I i, 0.1 ' 0.75 I.o L - Me:hionine (rnm) Fig. 6. Titration of methionyl-trna synthetase (0.33 pm) with L-methionine in the absene (0) and presene (A) of pyrophosphate (1.8 mm), in buffer A protein fluoresene, but no signifiant shift in the emission spetrum. D-Methionine has no disernible effet under the same onditions. The assoiation onstant determined from the saturation urve (Fig. 6) is 2.2 x lo4 M-l, a value in lose agreement 31 Eur. J. Biohem., Vol o L-Methionine (rnm) Fig.8. Titration of methionyl-trna synthetase (0.33 pm) by L-methwnine in the presene of various onentrations of ATP. ATP onn.: mm (0) ; mm (+) ; (A) and 3.0 mm (o), in buffer A with that derived from equilibrium dialysis performed under the same onditions. Furthermore, the assoiation onstant for methionine is not affeted by prior addition of pyrophosphate (1.8 mm). Binding of ATP to the Enzyme - Methionine Complex. The emission spetrum of methionyl-trna synthetase remains unaltered when ATP, AMP, (5 mm eah), Mg2+ (10 mm) or EDTA (1 mm) are added separately. However, in the presene of saturating onentrations of methionine, addition of ATP auses a marked dereases in fluoresene, whih levels off at /, of the initial enzyme fluoresene (Fig.7). This level is not affeted by addition of either Mg2+ (10 mm) or EDTA (1 mm) to the solution. Furthermore, an idential saturation urve for ATP was obtained when AMP (0.5 mm) was present in the reation mixture. The apparent assoiation onstant for ATP dedued from these experiments is 4 x lo4 M-l. In another series of experiments (Fig.8) in whih the order of addition of the substrates was inverted, the level of fluoresene attained in the presene of saturating amounts of

6 466 Binding Properties of Methionyl-tRNA Synthetase Eur. J. Biohem. Table 2. Apparent affinity onstant of methionyl-trna aynthetuse for pyrophmphate, in the presene of L-methimine and ATP, as measured by fluoresene spetrosopy lo-' x Apparent &. I-Methionine ATP for pyrophosphate mm mm M x In U 3 E %.- - d 100 ) o Pyrophosphate (mm) Fig.9. Titration of mthionyl-trna synthetase (0.2 pm) with sodium pyrophosphde in the presene of L-methionine (0.62 mm) and various Conentrations of ATP. ATP onn: 1.86mM (0); 0.62mM (A); 0.062mM (0) and 0.024mM (+), in buffer A. The initial levels of fluoresene at these onentrations of L-methionine and ATP were 83.5, 86.0, 96.5 and 105% of the fluoresene of the enzyme alone, respetively. Eah initial level has been normalized to 100 in the figure methionine was dependent on the onentration of ATP present, as expeted from the results in Fig.7. The apparent assoiation onstant for methionine (5 x lo4 M-I) was unaffeted when the ATP onentration was varied within the range of 0.1 to 3 mm. Binding of Pyrophosphate to Enzyme Saturated with Methionine and ATP. Pyrophosphate alone, tested at a onentration of 5 mm, does not affet the enzyme fluoresene. However, when added in the presene of saturating onentrations of methionine and ATP, it auses a marked inrease in fluoresene intnsity, from the initial level of 820/,, (see Fig.7) to a final level of 1120/,, whih is not affeted by the presene of Mg2+ (10 mm) or EDTA (1 mm). Table 2 shows the apparent assoiation onstant for pyrophosphate in the presene of a onstant and saturating amount of methionine and different onentrations of ATP. These values are derived from the titration urves presented in Fig. 9, in whih the initial levels of fluoresene in the absene of pyrophosphate were normalized to 1000/,. I ~-Methionyl adenylate (pm) Fig. 10. Titration of methionyl-trna synthetaae (0.26,uM in buffer E) with an aqueous solution of 5-methionyl adenyate at ph 3.7. Titration with equivalent amounts of the aqueous solution alone did not affet the ph of the buffered solution of enzyme and had no effet on the fluoresene emission Binding of Methionyl Adenylate to the Enzyme. Titration of methionyl-trna synthetase with methionyl adenylate prepared by hemial synthesis auses a pronouned quenhing of enzyme fluoresene, to a ha1 level of (Fig. lo), in ontrast to the enhaned fluoresene observed in the presene of methioninyl adenylate. These experiments were onduted at ph6.0, in view of the marked instability of methionyl adenylate at higher ph values. The same final level of fluoresene was also obtained at ph 7.6, but required higher onentrations of methionyl adenylate. The number of eight binding sites dedued from the titration urve presented in Fig. 10 is undoubtetdly overestimated, on aount of the relative instability of methionyl adenylate under the experimental onditions used. Binding of Analogues of Methionine to the Enzyme. The interation of methionyl-trna synthetase with two analogues of methionine whih lak the arboxyl funtion required for adenylate formation, was investigated by fluoresene spetrosopy. Saturating onentrations of L-methioninol and DL-3-methylthiopropylamine inrease the enzyme fluoresene intensity by 26 Ol0 and 12 Ole, respetively. Addition of saturating onentrations of ATP to these enzyme - ligand omplexes auses a further inrease of fluoresene from 112 to 125O/, in the ase of 3-methylthiopropylamine, and no signifiant hange in the ase of methioninol. The dissoiation onstants for methioninol and 3-methylthiopropylamine derived from these titration experiments are in lose agreement with their inhibition onstants Ki of 15pM

7 Vo1.24, No.3,1972 S. BLANQUET, G. FAYAT, J.-P. WALLER, and M. IWATSUBO 467 Ligand (LM) Fig.11. Variation of the rate onstant of the binding of L-mthioninyl adenylate or L-methionyl adenylate to methwnyltrna synthetase, a8 a funtion of the onentration of ligand. The rate onstants (l/t) were determined from first-order plots of the binding reation as measured by fluoresene in a stopped-flow apparatus. L-Methioninyl adenylate in buffer A (0); 0.2 ml of L-methionyl adenylate in water at ph 3.7 mixed with an equal volume of enzyme in buffer E (0) measured in the ATP-PPI exhange assay, where both behave as ompetitive inhibitors of methionine. IIINETIC MEASUREMENTS Reation of Methioninyl Adenylate with the Enzyme. The rate onstants of the interation of methioninyl adenylate with methionyl-trna synthetase were determined from first-order plots of the binding reation as measured by fluoresene in a stoppedflow apparatus. Fig. 11 shows that the rate onstant varies linearly with ligand onentration. Integration of the first order rate equation yields rate onstants of: 6 x lo6 M-l s-i and 1.8 s--l, orresponding to an apparent KB of 3 x lo6 M-l. Reation of Methionyl Adenylate with the Enzyme. Fig. 11 shows the rate onstants of the interation of methionyl adenylate with the enzyme, determined from first-order plots of the binding reations. The rate onstant varies linearly with ligand onentration. The alulated kineti assoiation and dissoiation onstants are 1.5 x los M-l s-l and 2 s-l respetively. The value for k, is underestimated, due to inevitable partial hydrolysis of methionyl adenylat late. The apparent affinity onstant KB is, therefore, higher than 0.8 x lo6 M-l. Reation of ATP with the Enzyme - Methionine Compkx. The binding of methionine to the enzyme is ompleted within dead time and therefore outside of the range of the stopped-flow equipment used. Similary, the binding of ATP to the enzyme 3-methylthiopropylamine omplex ould not be visualized. However, reation of ATP with the enzyme * methio- Sl* o 2.o ATP (mm) Fig. 12. Variation of the rate onstant of the binding of ATP to mthionyl-trna synthetase in the presene of a saturating amount of L-nzethionine (1 mm) as a funtion of onentration of ATP. Measurements were made in buffer A (+); in buffer A with 10 mm MgCI, (0) or with 0.1 mm EDTA (0) nine omplex ould be readily followed. Fig. 12 shows the variation of the apparent rate onstants (r-l), determined from first-order plots of the overall reation, as a funtion of ATP onentration. Measurements were performed in buffer A alone, as well as in buffer supplemented with magnesium or EDTA. As demonstrated infig. 12, the presene of magnesium auses a drasti (approximately 300-fold) inrease in the reation rates, over that observed in the presene of EDTA. The differene in reation rates observed when measurements were performed in buffer A alone or in the presene of EDTA an be asribed to a low, yet detetable level of ontaminatingmg2+ in buffer A. From atomi absorption spetrophotometri measurements performed as desribed in Methods, this ontamination was estimated at 0.7 pm, and the assoiation onstant of Mg2+ to the enzyme at 2 x lo4 M-l, assuming four idential subunits having one binding site eah. These values indiate that, under the onditions used for fluoresene measurements in buffer A in the absene of added Mg2+, a major proportion of the enzyme moleules must be free of magnesium. Reation of Pyropho<vphate with Enzyme 8attrated with Methionine and ATP. The rate onstants of the reation of pyrophosphate with the enzyme in the presene of saturating onentrations of methionine and ATP, determined from first-order plots of the binding reation, are shown in Fig.13. As in the previous experiment, whih dealt with the interation of ATP with the enzyme - methionine omplex (Fig.l2), the addition of magnesium ions greatly enhanes (approximately 100 times) the rate of the reation of pyrophosphate, over that measured in the presene of EDTA.

8 468 Binding Properties of Methionyl-tRNA Synthetase Eur. J. Biohem. "? Y ' 5 V r" -0 0, _ b 1 Pyrophosphate (mm) Fig. 13. Variation of the rate onstant of the binding of sodium pyrophosphute to methionyl-trna synthetase in the presene of L-methionine (1.0 mm) and ATP (1.0 mm), as a funtion of pyrophosphate onentration. Measurements were made in buffer A (+); in buffer A with 10 mm MgCI, (0) or with 0.1 mm EDTA (0) DISCUSSION Equilibrium dialysis experiments indiate that methionyl-trna synthetase, a tetrameri enzyme omposed of apparently idential subunits of moleular weight 43000, an bind four moleules of ATP or only two of methionine. The number of binding sites and the affinity onstants for these two ligands were unaffeted by the presene or absene of magnesium ions. Furthermore, the enzyme was shown to bind two moleules of methioninyl adenylate, measured by fluoresene and absorbane spetrosopy at equilibrium. In view of the strutural analogy of this ligand with methionyl adenylate [14], and their omparable kineti onstants as determined by the stopped-flow tehnique, we infer that only two moleules of methionyl adenylate an bind to the enzyme. The unexpeted finding that only two sites are oupied by methionine as well as methioninyl adenylate on the tetrameri enzyme, appears to indiate that only two out of four ATP moleules are impliated in adenylate formation under the onditions used. The two additional moleules of ATP may possibly be bound to two subunits of the enzyme whih are transiently onformationally bloked for adenylate formation, or else may serve an unidentified regulatory funtion. The former possibility implies a funtional assymmetry in the tetrameri enzyme, with only two methionyl adenylate moleules existing on the enzyme at a given time. Suh a mode of funtioning ould be related to a flip-flop type mehanism [ZO]. The harateristis of methionyl adenylate formation were examined by fluoresene spetrosopy. This approah is partiularly suitable for the visualization of the intermediary steps in the ativation reation atalyzed by methionyl-trna synthetase, sine most of the ligands involved give rise to harateristi and well-defined levels of enzyme fluoresene. When eah ligand is studied independently, the levels of fluoresene attained an be diretly related to enzyme-ligand omplex formation. In the presene of additional ligands, the fluoresene level observed is the weight average of the speifi fluoresenes of the different omplexed forms of the enzymes in dynami equilibrium. It is of partiular interest that interation of the enzyme with metliioninyl adenylate auses a 400/, inrease of the initial protein fluoresene, in ontrast to reation with methionyl adenylate whih lowers the initial level by 27 Ole. Thus, mere replaement of the methylene group in one ligand by a arbonyl group in the other profoundly affets enzyme fluoresene, most probably as a result of ligandindued onformational hanges, rather than through shielding of a neighbouring tryptophan residue. Interation of the enzyme with methioninol or 3-methylthiopropylamine, both of whih lak the arboxyl funtion, results in enhanement of fluoresene, although of lower magnitude than that observed to the presene of L-methionine. This onfirms earlier reports [14] on the non-essentiality of the arboxyl group for reognition of the amino aid. No variation in level of enzyme fluoresene is detetable in the presene of D-methionine, in aordane with the known stereospeifiity of the enzyme for L-methionine in the ativation reation [9]. It is also shown that addition of ATP to the enzyme * S-methyl-thiopropylamine omplex, whih is unable to reat with ATP to yield the orresponding adenylate, auses a further rise in fluoresene, indiating the formation of a ternary omplex. The level of fluoresene attained in the presene of both methionine and ATP is unaffeted by the presene of magnesium or EDTA. Sine the produt formed in the presene of magnesium ions is methionyl adenylate, we infer that this produt an be formed in the absene of magnesium. However, it should be noted that the fluoresene level attained in the presene of saturation amounts of methionine and ATP (Le. S20/0) differs from that obtained in the presene of hemially synthesized methionyl adenyllate alone (ie. 730/,). By analogy with the binding of two moleules of methioninyl adenylate, we assume that only two moleules of methionyl adenylate are bound to the enzyme under the latter onditions. On the other hand, in the experiment involving saturating amounts of methionine and ATP, the enzyme forms and retains two moleules of methionyl adenylate, and may, in addition, bind two more

9 Vo1.24, Ho.3, 1072 S. BLANQUET, G. FAYAT, J.-P. WALLER, and M. IWATSUBO 469 moleules of ATP, and possibly two of methionine in an unreative state at a given time. This explanation may aount for the differenes in the fluoresene levels observed in the two kinds of experiments. The existene of the enzyme methionyl adenylate omplex is further onfirmed by the fat that addition of saturating amounts of pyrophosphate to the enzyme saturated with ATP and methionine profoundly modifies the level of fluoresene, whih rises from 82O/, to 11Z0/,. The results reported in this work indiate that the presene of magnesium ions is not a prerequisite for interation of ATP or pyrophosphate with the enzyme, and for the formation of aminoayl adenylate and the reversal of this reation. However, rapjdmixing experiments reveal a dramati inrease in reation rates in the presene of Mg2+ both in the forward and in the reverse reation, whih an aount for the absolute dependene on Mg2+ observed in the ATP-PP, exhange reation. The finding that methionyl adenylate an be formed from ATP and methionine in the presene of EDTA, together with the observation from dialysis experiments that the enzyme an bind ATP under the same onditions, learly indiat,es that formation of the monomagnesium omplexes of ATP and PPi is not an absolute requirement for the reognition of ATP or pyrophosphate by methionyl-trna synthetase. A similar onlusion was reahed for reation of ATP with valyl-trna synthetase [7], while the opposite onlusion was drawn in the ase of isoleuyltrna synthetase, on the basis of kineti measurements alone [Zl]. The aelerating effet of magnesium on the formation of methi-onyl adenylate and on the reversal of this reation an be asribed to one of several possibilities. The simplest explanation is that interation of Mg2+ with ATP or PPi leads to a harge distribution more favorable to their interation with the enzyme. Alternatively, magnesium ions may interat diretly with the enzyme, induing a minor onformational hange whih is not detetable by fluoresene, or may diretly bind to the ative site of the enzyme, thereby modifying its harge distribution. In this onnetion, it should be realled that atomi spetrosopi determinations of the Mg2+ ontent of the enzyme has failed to show the presene of tightly bound metal, the affinity onstant for Mg2+ being of the order of 2 x lo4 M-l. A similar value was reported for the assoiation onstant of Mg2+ to valyl-trna synthetase [7]. The results reported learly indiate that fluoresene tehniques provide a partiularly valuable tool for monitoring the various reations atalyzed by methionyl-trna synthetase. The most striking finding emerging from this study is the demonstration of a dissymmetry in the subunits of methionyl-trna synthetase with respet to ligand-binding sites, whih reflets funtional assymmetry of the enzyme. Assessment of the signifiane of these observations in terms of the mehanism of reation will require the aquisition of additional information on the primary strutural relationship between the four subunits of the enzyme, as well as a detailed study of the role of trnamet in the reations atalyzed by methionyltrna synthetase. An extension of the present study along these lines should lead to the formulation of a funtional model for methionyl-trna synthetase. We are most grateful to Dr D. Pantaloni for stimulating disussions and advie throughout this work, to Dr A. di Frano for ritial reading of the manusript and to Dr D. Duval for aess to the atomi absorption spetrophotometer. REFERENCES 1. Loftfield, R. B., in Protein Synthesis (edited by E. 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Chem. 233 (1958) Silver, M. J., Rodalewiz, I., Douglas, Y., and Park, D., Anal. Biohem. 36 (1970) Myer, Y. P., and Shellman, J. A., Biohim. Biophys. Ata, 55 (1962) Bray, G. A., Anal. Biohemistry, 1 (1960) Iwatsubo, M., and Capeilkre, C., Biohim. Biophys. Ata, 146 (1967) Lazdunski, M., Petitler, C., Chappelet, D., and Lazdunski, C., Eur. J. Biohem. 20 (1971) Cole, F. X., and Shimmel, P. R., Biohemistry, 9 (1970) 481 and S. Blanquet, G. Fayat, and J.-P. Waller Laboratoire d'enzymologie du C.N.R.S. F-91 Gif-sur-Yvette, Frane M. Iwatsubo Laboratoire de G6nAtique Molhlaire du C.N.R.S, F-91 Gif-sur-Yvette, Frane