Eur. J. Biochem. 53,493-498 (1975)
Aminoacyl-tRNA Synthetases from Bacillus stearothermophilus. Asymmetry of Substrate Binding to Tyrosyl-tRNA Synthetase Hans Rudolf BOSSHARD, Gordon L. E. KOCH, and Brian S. HARTLEY Medical Research Council Laboratory of Molecular Biology, Cambridge (Received August 13, 1974/January 14, 1975)
The interaction of L-tyrosine, L-tyrosyladenylate and tRNATyrwith tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by equilibrium dialysis, gel filtration and fluorescence spectroscopy. The enzyme, which consists of two identical subunits (mol. wt 2 x 44000), binds only a single molecule of L-tyrosine per dimer with a Kd of 2 x M at pH 7.8 and 23 "C. The tyrosyltRNA synthetase - tyrosyladenylate complex which was isolated by gel filtration also has one adenylate bound per dimeric enzyme molecule. In contrast, two tRNATyrmolecules bind per enzyme dimer, but the two binding sites are not equivalent having Kd values of 2 x l o p 7 M and 1.3 x M respectively at pH 6.5 and 25 "C. Since crystallographic analysis of the free enzyme [2] shows that the monomer is the asymmetric unit, the data indicate that substrate binding induces asymmetry in the enzyme.
Tyrosyl-tRNA synthetase from Bacillus stearothermophilus is a dimeric enzyme composed of two identical subunits of mol. wt 44000 [l]. Preliminary X-ray data show that the monomer is the asymmetric unit of the molecule [2] (D. M. Blow and coworkers, personal communication). As regards molecular weight and subunit composition this enzyme is similar to several other synthetases which all are of dimeric structure with monomers of between about 40000 and SO000 mol. wt [3]. For this class of synthetase as well as for others there seems to be no common relation between the number of substrate-binding sites or active sites and the subunit composition. Reports range from the simple pattern of one site per subunit to one per tetramer [4]. In addition, some conflicting data on the number of substrate binding sites for individual enzymes are in the literature, e.g. one and two binding sites, respectively, were found for amino acid and tRNA in the case of the serine and tyrosine activating enzymes from Escherichia coii [S-8]. Although in these two particular cases the discrepancies are probably due to the analytical .
Dcfinnirion. One A,,, unit is the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a I-cm light path. Enzymes. Tyrosyl-tRNA synthetase o r L-tyrosine : t R N A ligase (AMP-forming) (EC 6.1.1.1).
Eur. J. Biochem. 53 (1975)
methods employed in quantitating enzyme-ligand complexes, they could nevertheless indicate nonequivalence of the sites. In this report we present binding studies with the tyrosyl-tRNA synthetase from Bacillus stearothermophilus. The results show that this enzyme has one strong binding site per dimer for L-tyrosine, L-tyrosyladenylate and tRNATy' plus one additional weak binding site for a second tRNA molecule. Together with the crystallographic evidence [2] (D. M. Blow and coworkers, personal communication) this adds up to a conflicting situation which can be reconciled by assuming that substrate binding induces asymmetry upon the enzyme molecule giving rise to the phenomenon of half-of-sites reactivity [9]. MATERIALS AND METHODS Materials
Isolation and purification of tyrosyl-tRNA synthetase has been described [l]. Crude tRNA from B. stearothermophilus was isolated as described by Littauer et al. [lo]. Purification of the tyrosineaccepting species was accomplished by consecutive chromatography on benzoylated DEAE-cellulose and on the reversed phased chromatography system
494
RPC5 [l I]. The benzoylated DEAE-cellulose column was first eluted with a gradient of 0.2 to 1.3 M NaCl in 10 mM MgCI2/0.05 M sodium acetate pH 5.0 and afterwards with a gradient from 0 to 20% ethanol/ 1.3 M NaCl in the same buffer. The tyrosine-accepting fractions eluted between 3 ”/, and 6 ”/, ethanol in the second gradient. This material was rechromatographed twice on the reversed phase chromatography system. For the first rechromatography a gradient from 0.5 to 0.9 M NaCl in the above acetate buffer was used. The fractions eluting around 0.68 M NaCl were rerun with a gradient from 0.6 to 0.8 M NaCl. The final material had a tyrosine acceptance of 1.2 nmol per unit and was assumed to be 75 pure. The partly enriched fraction of tRNAL‘” was eluted from the benzoylated DEAE-cellulose column at 0.75 M NaCl in the first gradient, with a leucine acceptance of 0.4 nmol per AZ6,,unit. A fraction enriched in tRNATyrfrom E. coli was kindly provided by R. Coulson. Further purification was accomplished by chromatography on the reversed phase chromatography system to an acceptance of 1.3 nmol of tyrosine per A260 unit in the heterologous charging assay with tyrosyl-tRNA synthetase from B. stearothrrmophilus. Inorganic pyrophosphatase was from Worthington Biochemical Company (Freehold, N.J., U.S.A.). 1 pl of the preparation containing 1.2 mg/ml cleaved 1 pmol of pyrophosphate per min. 14C- and 3Hlabelled L-tyrosine and [I4C]ATPand [y-32P]ATPwere from the Radiochemical Centre (Amersham, U.K.). The purity of L-tyrosine was checked by high voltage ionophoresis at pH 2.2 and by paper chromatography in the system n-butanol/acetic acid/water/ pyridine(l5:3: 12: 10). ThepurityofATP waschecked by thin-layer chromatography on polyethylenimine coated plastic sheets (Polygram CEL 300 PEI, Machery and Nagel, Diiren, W. Germany) using 0.5 M phosphate pH 3.5 as mobile phase 1121. M e thodLs Procedures for pyrophosphate-ATP exchange assay, tRNA charging and amino acid analysis have been given elsewhere [I]. Equ ilihrium Dialysis
Apparatus and details of the experimental procedure were as described previously [13]. The buffer was 0.144 M Tris/HCl pH 7.9 (25 “C) containing 10mM MgCI,, 10mM 2-mercaptoethanol and 0.1 mM phenylmethanesulfonylfluoride. Equilibration was performed at room temperature (22 - 24 “C) for 24 h. Membranes were cut from Visking dialysis tubing
Aminoacyl-t RNA Synthetases from Bacillus steurorhrrmophilu.s
18/32, boiled in 0.1 M Na,CO, and in 1 mM ethylenediaminetetraacetic acid and rinsed with distilled water. Before use they were soaked in the Tris buffer for at least 3 h at room temperature. Preparation and Isolation the Aminoacyladenylatr-Enzyme Complex
of
Formation of the complex was accomplished by adding enzyme stock solutions in 0.144 M Tris/HCl, pH 7.9 (25 ‘C), 10 mM MgCl,, 10 mM 2-mercaptoethanol to 3H-labelled L-tyrosine, 14C- or (y3,P)labelled ATP and inorganic pyrophosphatase, all in the same Tris buffer. Final concentrations were usually in the range of 5- 105 pM for enzyme, 300600 pM for ATP and amino acid and 15 pg/ml for pyrophosphatase. The reaction mixtures (150 - 250 pl) were incubated usually for 2 min at 20 C, chilled in ice and applied immediately to a Sephadex G-25 column (0.6 x 26 cm) run at 4 “C with the same Tris buffer used in the reaction mixture minus enzyme and substrates. Fractions of 10 drops (about 0.62 ml) were collected at a rate of 12- 15 ml/h. Aliquots of 50 pl of each fraction were counted in 4 ml of scintillation liquid (60 g naphthalene, 4 g 2,5-diphenyloxazole, 0.2 g 1,4-di[2-(5-phenyloxazolyl)]benzene, 100 ml methanol, ethyleneglycol and dioxane to a final volume of 11). Appropriate aliquots of peak fractions were also used to determine enzyme concentrations by amino acid analysis and absorbance at 280 nm. Fluorescence Titrutions
Fluorescence measurements were performed using a Perkin-Elmer spectrofluorimeter model MPF 3 at 25 _+ 0.2 “C in 0.1 M cacodylate buffer pH 6.5 and in the presence of 10 mM MgCI, unless otherwise stated. The enzyme solution (200 pl) was placed in a microfluorescence cuvette (3 mm light path) and titrated stepwise with tRNA added in small quantities with a Hamilton microsyringe. The total volume increase was 10% and was corrected for. Excitation was at 290 nm at a band width of 2- 3 nm. No ultraviolet damage of the enzyme was detected using this narrow slit on the excitation side. Emission was recorded at 340 nm and at a band width of between 8 and 16 nm. Stability of the arc and photomultiplier were checked by measuring a control solution of free enzyme between experimental readings. Since the excitation spectrum of the enzyme and the absorption spectrum of tRNA overlap the total absorption at the excitation wavelength increased during the titration. Therefore the measured fluorescence intensity had to be corrected according to Eur. J. Biochem. 53 (1975)
495
H. R. Bosshard, G. L. E. Koch, and B. S. Hartley
Fcorrected - Fmeasured x C. The correction factor C was
takenfromref. [ 1 4 ] a s C = ( k / k , ) ~ ( l - e - ~ ~ ~ ) / ( l - e - ~ ~ ) where k = molar absorption coefficient x molar concentration and d = length of light path in the direction of the excitation beam. The index 0 refers to the pure enzyme solution. Use of this formula was justified [14] since the total absorbance A (= kd/2.3) at 290 nm was below 0.01 for the highest concentration of enzyme alone and below 0.2 at the highest tRNA concentration.
I
\ \
RESULTS The Enzyme- Tyrosine Complex
The stoichiometry and dissociation constant for the binding of L-tyrosine to the free enzyme were determined by equilibrium dialysis. One molecule of amino acid was found to bind per dimeric enzyme molecule with a Kd of 2 x l o p 5 M at 23 k 1 "C and at pH 7.8 ( I = 0.1) (Fig. 1). No loss of enzyme activity occurred during a 24 h experiment as judged from unchanged pyrophosphate-ATP exchange activity. The Aminoucyludenylute-Enzyme Complex
The complex isolated by gel-chromatography had an average stoichiometry of 0.97 tyrosyladenylate molecules per enzyme dimer and the values of individual experiments ranged from 0.75 to 1.1 (Fig.2). This stoichiometry was independent of enzyme concentration in the incubation mixture between 9 pM and 105 pM at constant ligand concentrations of 0.35 mM ATP and 0.39 mM L-tyrosine. The stoichiometry also did not change when ATP was increased from 0.35 mM to 2 mM and L-tyrosine from 0.39 mM to 1 mM at 54 pM enzyme. In addition the stoichiometry was not influenced by the time of incubation of the enzyme with substrates in the range of 30 s to 12 min at 20 "C. Incubation for 2 min and subsequent storage at 4 "C for 2 h before gel chromatography had no influence on the number of adenylate binding sites. All these observations indicate that the enzyme was saturated with tyrosyladenylate under the chosen conditions. In the absence of pyrophosphatase only about 0.5 mol of tyrosyladenylate could be isolated per mol of enzyme. Presumably, any back reaction in the direction of pyrophosphate incorporation into adenylate with subsequent formation of ATP was suppressed by the action of inorganic pyrophosphatase. The stability at 4 "C of the isolated complex was checked by rechromatography after different periods of storage. An apparent half-life of more than 20 h at pH 7.8 was found. In order to show that the radioactivity in the complex originating from [I4C]ATP Eur. J. Biochem. 53 (1975)
r
Fig. 1. Scatchard plot f o r the binding of L-tyrosine to tyrosyl-tRNA synthetasr. According to r / c = K(n - r ) where r = moles of amino acid bound per mole of enzyme, c = moles of free amino acid, n = number of independent binding sites and K = association constant
12
t
=' 10 E
. 0
g 8 c
c
0
E
4
6
6 ' :
I
I
2
2
0
n
n
"2
6
10 14 18 Fraction numbers
22
26
.
Fig. 2. Isolation of the tyrosyl-tRNA synthetasr- tyrosylaclenylate complex by gel filtration. In the above experiment enzyme and substrates were incubated for 2.5 min at 18 "C in a total volume of 0.26 ml. Final concentrations were 0.35 mM [14C]ATP,0.39 mM ~-[~H]tyrosine and 54 pM enzyme. I n a parallel experiment 0.3 mM [Y-~'P]ATPwas used instead of [14C]ATP.(0)Enzyme from amino acid analysis (fractions 7 - 9) and absorbance at 280 nm (1 mg per ml equals 1.3 A-units per cm); (A) AMP and ATP, respectively; (0)L-tyrosine; (0)
496
Aminoacyl-tRNA Synthetases from Bacillus stearotherrnophilus
was really attributable to AMP in the tyrosyladenylate and not to a complex of ATP with enzyme, [ Y - ~ ~ P ] ATP was used in one experiment. All 32P-radioactivity eluted with the free ATP (Fig. 2). The possibility that tyrosyl-tRNA synthetase con0.0 ** tains two potential and equivalent active sites but IIP ; 0.7 \ **. that only one at a time may be occupied, i.e. that the second site can form adenylate only after the first 0.6 site has dissociated from its adenylate, was tested as 0.5 follows. Tyrosyladenylate-enzyme complex was prepared in the usual manner, isolated by gel filtration and incubated at a concentration of 5 pM with 0 2 4 6 8 10 12 200 pM ATP and 228 pM ~ - [ ~ H ] t y r o s i nfor e 2 min [ tRNAl( P.M ) at 18 "C and rechromatographed immediately. Of the Fig. 3. Fluorescence titrution of tyrosyl-tRNA synthetaw (2.7 p M ) recovered tyrosyladenylate-enzyme complex, 25 % had with tRNAs. (0) tRNATy' (1.2 nmol tyrosine acceptance per A,,, ~ - [ ~ H ] t y r o s i nincorporated. e This corresponds to a unit); (0) partly enriched tRNALeU(0.4 nmol leucine acceptance per A,,, unit); 0.1 M cacodylate pH 6.5, 10 mM MgCI,, 25 'C 15 % net incorporation of labelled tyrosine after correcting for the free enzyme formed by decomposition of the unlabelled complex for the duration of the experiment. In other words, the rate of formation of adenylate is reduced to the order of lop4 to s-l whereas the rate of de novo synthesis catalysed by the free enzyme is 18 s-' (A. R. Fersht, personal com125 150 munication).
\.
1
The Enzyme-tRNA Complex The fluorescence of tyrosyl-tRNA synthetase is reduced by 50 "/, upon saturation with tRNATyrfrom B . stearothermophilus (Fig. 3). No quenching was observed in the presence of a mixture of tRNA partially enriched in tRNAL2"but devoid of tyrosine acceptance activity. Analysis of the titration curve in Fig.3 by extrapolating the initial slope to the extrapolated terminal base line at high tRNA concentration [15] gives a stoichiometry of 1.9 tRNA binding sites per enzyme molecule. Collection of data at lower enzyme concentration revealed that the two sites are not equivalent (Fig. 4). The degree of fluorescence quenching is approximately 25 % for each site. The dissociation constants could be estimated (but not accurately determined, see [ 161) from experiments performed at enzyme concentrations below the first Kd and in between the two K,-values. The values at pH 6.5 (0.1 M cacodylate), 25 "C and in the presence of 10 mM MgCl, are 2 x lo-' M or less for the stronger site and around 1.3 x lop6 M for the weaker. Decreasing the pH to 5.4 (0.1 M sodium acetate) did not change the Kd-values within the limit of error which is, however, rather broad. But at pH 7.8 (0.144 M Tris/HCl) binding to both sites is weaker with Kdvalues of 0.7 x M and 5 x l o p 6 M. tRNA binding is independent of magnesium ions and Kd-values are unchanged between 0 and 1 5 m M MgCl, but decrease by a factor of 2 to 3 at 50 mM MgC1,.
.
"0
10
* .*:
x) Fo-F
.... 30
40
Fig.4. Scatchard plot f o r the binding of' tRNA' y' to tyrosyl-tRNA synrherase. F, is the fluorescence (arbitrary units) in the absence, and F in the presence of tRNATyr;enzyme (0.15 pM) in a 0.1 M cacodylate pH 6.5, 10 mM MgCI,, 25 "C. The limiting slopes drawn correspond to dissociation constants, K d ,of 0.14 and 1.25 x lo-' M. These values were determined in parallel experiments at 0.05 and 0.4 pM enzyme concentrations, respectively
Binding of tRNATyrfrom E. coli to the B. stearothermophilus enzyme exhibits the same two nonequivalent sites with similar affinities to those observed with the homologous system (data not shown).
DISCUSSION Before considering the implications of the results obtained the reliability of the data needs assessment, since binding studies on many similar enzymes have Eur. J. Biochem. 53 (1975)
14
H. R. Bosshard, G. L. E. Koch, and B. S. Hartley
been bedevilled by artefact. One of the main sources of error is inaccurate estimates of the concentration of active enzyme. We have determined the amount of enzyme protein in samples by direct amino acid analysis of hydrolysates and are confident that the error is 5%. However, this does not provide an estimate of the proportion of active species in the preparation. Since the preparation of enzyme used in this study was homogeneous by disc-gel electrophoresis, had the highest specific activity of seven independent preparations and crystallised easily, we have no reason to believe it contained significant amounts of inactive material. The binding of the small ligand tyrosine was measured by equilibrium dialysis and artefacts due to the Donnan effect or by adsorption to the membrane were not significant in our experiments. Stoichiometries were obtained from studies at high enzyme concentrations to enhance the reliability of the estimates. The binding of tRNA to the enzyme could not be measured by dialysis and fluorescence quenching had to be used for this purpose. The main problem with this technique is the ultraviolet absorption of the nucleic acid which must be corrected for. The formula derived by Ehrenberg et al. [14] was used for this correction since the total absorbances of enzyme and tRNA were within the prescribed limits. The other complication arose from the presence of more than one type of binding site for tRNA on the enzyme. For this reason estimates of the two dissociation constants could only be approximated. Use of gel filtration for studying the binding of aminoacyladenylate has been criticised on the grounds that the complex is not in equilibrium with the substrates throughout the isolation and would therefore yield under-estimates of the true stoichiometry. We have measured the rate of decomposition of the isolated enzyme-adenylate complex under the conditions of the isolation procedure and found it to have a half-life of over 20 h. Consequently it is unlikely that this factor affects the results of the experiment which lasts only about 20 min. Saturation of the available adenylate binding sites was evident from the independence of the measured stoichiometries on the enzyme and substrate concentrations used. The main source of error we have encountered was the high affinity of the enzyme for pyrophosphate. In the absence of pyrophosphatase only about 0.5 mol of aminoacyladenylate could be detected per mol of enzyme whereas this was doubled upon addition of pyrophosphatase. Further additions of pyrophosphatase did not increase the stoichiometry indicating that this source of error had been fully eliminated. Hence it is unlikely that the binding stoichiometries of tyrosine, tRNA and tyrosyladanylate to tyrosylEur. J. Biochem. 53 (1975)
497
tRNA synthetase are grossly incorrect. It is therefore significant that all these ligands manifest some asymmetry in their binding to the dimeric enzyme. Tyrosine and tyrosyladenylate bind to only one site on the enzyme under the conditions used. tRNATy‘has two binding sites on the enzyme but the affinities differ by almost one order of magnitude. This general asymmetry is somewhat surprising in view of the high degree of symmetry of the enzyme molecule in the crystal [ 2 ] . Assuming that this symmetry holds for the dimer in solution, one must conclude either that there is steric overlap between two adjacent binding sites or that substrate binding induces conformational asymmetry. There is support for the latter hypothesis from studies of the reaction of the native enzyme with 5,5’-dithio-bis-(2-nitrobenzoicacid) [l]. Of the four cysteine residues in the dimer only one reacted with the reagent and reaction was slow. Steric hindrance between two adjacent but relatively inaccessible thiol groups in a rigid dimer is again a possibility but stretches coincidence. An alternative assumption is of a major symmetrical conformation lacking ligand binding sites in equilibrium with a minor asymmetric form with a single ligand binding site. The thiol reactivity would be explained if the symmetrical form had four “buried” thiol groups and the asymmetric form had three “buried” and one “free” thiol groups. A similar explanation was adduced to explain the change in thiol reactivity of methionyl-tRNA synthetase of E. coli consequent on aminoacyladenylate formation [13]. Evidence is beginning to accumulate for functional and structural asymmetry in many synthetases and the point has been reached where some explanation for this phenomenon will have to be sought. One possibility is that the flip-flop type mechanism proposed by Harada and Wolfe [17] and Lazdunski et al. [18] operates in such cases. We have attempted to test this by examining the incorporation of [3H]tyrosine and ATP into the isolated enzyme-adenylate complex. If a flip-flop type process based on two equivalent active sites is operative we would expect the radioactivity to be incorporated into the complex at a rate equivalent to de n o w synthesis. The results obtained show that although there is some incorporation of radioactivity the rate is much slower than that observed for de novo synthesis. Thus a simple flip-flop mechanism is unlikely. Although two equivalent tyrosine and adenylate binding sites (or active sites, for the sake of argument) must be excluded from the available data, there is still the possibility that a second weak site escaped detection by our experimental approach. There is independent kinetic evidence for such a weak site
498
H. R. Bosshard, G. L. E. Koch, and B. S. Hartley: Aminoacyl-tRNA Synthetases from Bacillus stearoihermophilus
which forms but never accumulates tyrosyladenylate since its rate of hydrolysis (or dissociation) is faster than its rate of formation (A. Fersht and coworkers, unpublished). The existence of such a second weak site would, of course, be consistent with the asymmetric model of the functional enzyme deduced from our data. It will be most important to know whether this model has any significance for the catalytic specificity of tyrosyl-tRNA synthetase. H.R.B. is the recipient of a fellowship from the Schweizerischer Naiionaljonds.
REFERENCES 1. Koch, G . L. E. (1974) Biochemistry, 13, 2307-2312. 2. Reid, B. R., Koch, G. L. E., Boulanger, Y., Hartley, B. S. & Blow, D. M. (1973) J . Mol. Biol. 80, 199-201. 3. Hartley, B. S. (1974) Symp. SOC.Gen. Microbiol. 24, 151-182. 4. Loftfield, R. B. (1972) Progr. Nucfeic Acid Res. Mol. Biol. 12, 87- 128. 5. Knowles, J . R., Katze, J. R., Konigsberg, W. & SOH, D. (1970) J . Biol. Chem. 245, 1407- 1415.
6. Boecker, E. A. & Cantoni, G. L. (1973) Biochemisir.r., 12, 2384-2389. 7. Krajewska-Grynkiewicz, K., Buonocore, V. & Schlesinger, S. (1973) Biochim. Biophys. Acta, 312, 518-527. 8. Chousterman, S. & Chapeville, F. (1973) Eur. J . Biochem. 35, 51-56. 9. Levitzki, A., Stallcup, W. & Koshland, D. E., Jr (1971) Biochemistry, 10, 3371 - 3378. 10. Littauer, U. Z . , Yanofsky, S. A., Novogrodsky, A., Bursztyn, H., Galanter, Y . & Katchalski, E. (1969) Biochim. Biophys. Acta, 195, 29 - 49. 11. Pearson, R. L., Weiss, J. F. & Kelmers, A . D. (1971) Biochim. Biophys. Acta, 228, 770- 774. 12. Randerath, K. & Randerath, E. (1964) J . Chromatogr. 16, 111 - 125. 13. Bruton, C. J. & Hartley, B. S. (1970) J . Mol. Biol. 52, 165- 178. 14. Ehrenberg, M., Cronvall, E. & Rigler, R. (1971) FEBSLett. 18, 199- 203. 15. Velick, S. F., Parker, C. W. & Eisen, H. N. (1960) Proc. Nut1 Acad. Sci. U . S . A . 46, 1470-1482. 16. Weder, H. G., Schildknecht, T., Lutz, R. A. & Kesselring, P. (1974) Eur. J . Biochem. 42, 475-481. 17. Harada, K. & Wolfe, R. G . (1968) J . Bio/. C/zem. 243, 41314137. 18. Lazdunski, C., Petitclerc, C., Chappelet, D. & Lazdunski, C. (1971) Eur. J . Biochem. 20, 124- 139.
H. R. Bosshard and G . L. E. Koch, M.R.C. Laboratory of Molecular Biology, Postgraduate Medical School, University of Cambridge, Hills Road, Cambridge, Great Britain, CB2 2QH B. S. Hartley, Department of Biochemistry, Imperial College of Science and Technology, Prince-Consort Road, London, Great Britain, SW7 2AY
Eur. J. Biochem. 53 (1975)
Aminoacyl-tRNA Synthetases from Bacillus stearothermophilus. Asymmetry of Substrate Binding to Tyrosyl-tRNA Synthetase Hans Rudolf BOSSHARD, Gordon L. E. KOCH, and Brian S. HARTLEY Medical Research Council Laboratory of Molecular Biology, Cambridge (Received August 13, 1974/January 14, 1975)
The interaction of L-tyrosine, L-tyrosyladenylate and tRNATyrwith tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by equilibrium dialysis, gel filtration and fluorescence spectroscopy. The enzyme, which consists of two identical subunits (mol. wt 2 x 44000), binds only a single molecule of L-tyrosine per dimer with a Kd of 2 x M at pH 7.8 and 23 "C. The tyrosyltRNA synthetase - tyrosyladenylate complex which was isolated by gel filtration also has one adenylate bound per dimeric enzyme molecule. In contrast, two tRNATyrmolecules bind per enzyme dimer, but the two binding sites are not equivalent having Kd values of 2 x l o p 7 M and 1.3 x M respectively at pH 6.5 and 25 "C. Since crystallographic analysis of the free enzyme [2] shows that the monomer is the asymmetric unit, the data indicate that substrate binding induces asymmetry in the enzyme.
Tyrosyl-tRNA synthetase from Bacillus stearothermophilus is a dimeric enzyme composed of two identical subunits of mol. wt 44000 [l]. Preliminary X-ray data show that the monomer is the asymmetric unit of the molecule [2] (D. M. Blow and coworkers, personal communication). As regards molecular weight and subunit composition this enzyme is similar to several other synthetases which all are of dimeric structure with monomers of between about 40000 and SO000 mol. wt [3]. For this class of synthetase as well as for others there seems to be no common relation between the number of substrate-binding sites or active sites and the subunit composition. Reports range from the simple pattern of one site per subunit to one per tetramer [4]. In addition, some conflicting data on the number of substrate binding sites for individual enzymes are in the literature, e.g. one and two binding sites, respectively, were found for amino acid and tRNA in the case of the serine and tyrosine activating enzymes from Escherichia coii [S-8]. Although in these two particular cases the discrepancies are probably due to the analytical .
Dcfinnirion. One A,,, unit is the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a I-cm light path. Enzymes. Tyrosyl-tRNA synthetase o r L-tyrosine : t R N A ligase (AMP-forming) (EC 6.1.1.1).
Eur. J. Biochem. 53 (1975)
methods employed in quantitating enzyme-ligand complexes, they could nevertheless indicate nonequivalence of the sites. In this report we present binding studies with the tyrosyl-tRNA synthetase from Bacillus stearothermophilus. The results show that this enzyme has one strong binding site per dimer for L-tyrosine, L-tyrosyladenylate and tRNATy' plus one additional weak binding site for a second tRNA molecule. Together with the crystallographic evidence [2] (D. M. Blow and coworkers, personal communication) this adds up to a conflicting situation which can be reconciled by assuming that substrate binding induces asymmetry upon the enzyme molecule giving rise to the phenomenon of half-of-sites reactivity [9]. MATERIALS AND METHODS Materials
Isolation and purification of tyrosyl-tRNA synthetase has been described [l]. Crude tRNA from B. stearothermophilus was isolated as described by Littauer et al. [lo]. Purification of the tyrosineaccepting species was accomplished by consecutive chromatography on benzoylated DEAE-cellulose and on the reversed phased chromatography system
494
RPC5 [l I]. The benzoylated DEAE-cellulose column was first eluted with a gradient of 0.2 to 1.3 M NaCl in 10 mM MgCI2/0.05 M sodium acetate pH 5.0 and afterwards with a gradient from 0 to 20% ethanol/ 1.3 M NaCl in the same buffer. The tyrosine-accepting fractions eluted between 3 ”/, and 6 ”/, ethanol in the second gradient. This material was rechromatographed twice on the reversed phase chromatography system. For the first rechromatography a gradient from 0.5 to 0.9 M NaCl in the above acetate buffer was used. The fractions eluting around 0.68 M NaCl were rerun with a gradient from 0.6 to 0.8 M NaCl. The final material had a tyrosine acceptance of 1.2 nmol per unit and was assumed to be 75 pure. The partly enriched fraction of tRNAL‘” was eluted from the benzoylated DEAE-cellulose column at 0.75 M NaCl in the first gradient, with a leucine acceptance of 0.4 nmol per AZ6,,unit. A fraction enriched in tRNATyrfrom E. coli was kindly provided by R. Coulson. Further purification was accomplished by chromatography on the reversed phase chromatography system to an acceptance of 1.3 nmol of tyrosine per A260 unit in the heterologous charging assay with tyrosyl-tRNA synthetase from B. stearothrrmophilus. Inorganic pyrophosphatase was from Worthington Biochemical Company (Freehold, N.J., U.S.A.). 1 pl of the preparation containing 1.2 mg/ml cleaved 1 pmol of pyrophosphate per min. 14C- and 3Hlabelled L-tyrosine and [I4C]ATPand [y-32P]ATPwere from the Radiochemical Centre (Amersham, U.K.). The purity of L-tyrosine was checked by high voltage ionophoresis at pH 2.2 and by paper chromatography in the system n-butanol/acetic acid/water/ pyridine(l5:3: 12: 10). ThepurityofATP waschecked by thin-layer chromatography on polyethylenimine coated plastic sheets (Polygram CEL 300 PEI, Machery and Nagel, Diiren, W. Germany) using 0.5 M phosphate pH 3.5 as mobile phase 1121. M e thodLs Procedures for pyrophosphate-ATP exchange assay, tRNA charging and amino acid analysis have been given elsewhere [I]. Equ ilihrium Dialysis
Apparatus and details of the experimental procedure were as described previously [13]. The buffer was 0.144 M Tris/HCl pH 7.9 (25 “C) containing 10mM MgCI,, 10mM 2-mercaptoethanol and 0.1 mM phenylmethanesulfonylfluoride. Equilibration was performed at room temperature (22 - 24 “C) for 24 h. Membranes were cut from Visking dialysis tubing
Aminoacyl-t RNA Synthetases from Bacillus steurorhrrmophilu.s
18/32, boiled in 0.1 M Na,CO, and in 1 mM ethylenediaminetetraacetic acid and rinsed with distilled water. Before use they were soaked in the Tris buffer for at least 3 h at room temperature. Preparation and Isolation the Aminoacyladenylatr-Enzyme Complex
of
Formation of the complex was accomplished by adding enzyme stock solutions in 0.144 M Tris/HCl, pH 7.9 (25 ‘C), 10 mM MgCl,, 10 mM 2-mercaptoethanol to 3H-labelled L-tyrosine, 14C- or (y3,P)labelled ATP and inorganic pyrophosphatase, all in the same Tris buffer. Final concentrations were usually in the range of 5- 105 pM for enzyme, 300600 pM for ATP and amino acid and 15 pg/ml for pyrophosphatase. The reaction mixtures (150 - 250 pl) were incubated usually for 2 min at 20 C, chilled in ice and applied immediately to a Sephadex G-25 column (0.6 x 26 cm) run at 4 “C with the same Tris buffer used in the reaction mixture minus enzyme and substrates. Fractions of 10 drops (about 0.62 ml) were collected at a rate of 12- 15 ml/h. Aliquots of 50 pl of each fraction were counted in 4 ml of scintillation liquid (60 g naphthalene, 4 g 2,5-diphenyloxazole, 0.2 g 1,4-di[2-(5-phenyloxazolyl)]benzene, 100 ml methanol, ethyleneglycol and dioxane to a final volume of 11). Appropriate aliquots of peak fractions were also used to determine enzyme concentrations by amino acid analysis and absorbance at 280 nm. Fluorescence Titrutions
Fluorescence measurements were performed using a Perkin-Elmer spectrofluorimeter model MPF 3 at 25 _+ 0.2 “C in 0.1 M cacodylate buffer pH 6.5 and in the presence of 10 mM MgCI, unless otherwise stated. The enzyme solution (200 pl) was placed in a microfluorescence cuvette (3 mm light path) and titrated stepwise with tRNA added in small quantities with a Hamilton microsyringe. The total volume increase was 10% and was corrected for. Excitation was at 290 nm at a band width of 2- 3 nm. No ultraviolet damage of the enzyme was detected using this narrow slit on the excitation side. Emission was recorded at 340 nm and at a band width of between 8 and 16 nm. Stability of the arc and photomultiplier were checked by measuring a control solution of free enzyme between experimental readings. Since the excitation spectrum of the enzyme and the absorption spectrum of tRNA overlap the total absorption at the excitation wavelength increased during the titration. Therefore the measured fluorescence intensity had to be corrected according to Eur. J. Biochem. 53 (1975)
495
H. R. Bosshard, G. L. E. Koch, and B. S. Hartley
Fcorrected - Fmeasured x C. The correction factor C was
takenfromref. [ 1 4 ] a s C = ( k / k , ) ~ ( l - e - ~ ~ ~ ) / ( l - e - ~ ~ ) where k = molar absorption coefficient x molar concentration and d = length of light path in the direction of the excitation beam. The index 0 refers to the pure enzyme solution. Use of this formula was justified [14] since the total absorbance A (= kd/2.3) at 290 nm was below 0.01 for the highest concentration of enzyme alone and below 0.2 at the highest tRNA concentration.
I
\ \
RESULTS The Enzyme- Tyrosine Complex
The stoichiometry and dissociation constant for the binding of L-tyrosine to the free enzyme were determined by equilibrium dialysis. One molecule of amino acid was found to bind per dimeric enzyme molecule with a Kd of 2 x l o p 5 M at 23 k 1 "C and at pH 7.8 ( I = 0.1) (Fig. 1). No loss of enzyme activity occurred during a 24 h experiment as judged from unchanged pyrophosphate-ATP exchange activity. The Aminoucyludenylute-Enzyme Complex
The complex isolated by gel-chromatography had an average stoichiometry of 0.97 tyrosyladenylate molecules per enzyme dimer and the values of individual experiments ranged from 0.75 to 1.1 (Fig.2). This stoichiometry was independent of enzyme concentration in the incubation mixture between 9 pM and 105 pM at constant ligand concentrations of 0.35 mM ATP and 0.39 mM L-tyrosine. The stoichiometry also did not change when ATP was increased from 0.35 mM to 2 mM and L-tyrosine from 0.39 mM to 1 mM at 54 pM enzyme. In addition the stoichiometry was not influenced by the time of incubation of the enzyme with substrates in the range of 30 s to 12 min at 20 "C. Incubation for 2 min and subsequent storage at 4 "C for 2 h before gel chromatography had no influence on the number of adenylate binding sites. All these observations indicate that the enzyme was saturated with tyrosyladenylate under the chosen conditions. In the absence of pyrophosphatase only about 0.5 mol of tyrosyladenylate could be isolated per mol of enzyme. Presumably, any back reaction in the direction of pyrophosphate incorporation into adenylate with subsequent formation of ATP was suppressed by the action of inorganic pyrophosphatase. The stability at 4 "C of the isolated complex was checked by rechromatography after different periods of storage. An apparent half-life of more than 20 h at pH 7.8 was found. In order to show that the radioactivity in the complex originating from [I4C]ATP Eur. J. Biochem. 53 (1975)
r
Fig. 1. Scatchard plot f o r the binding of L-tyrosine to tyrosyl-tRNA synthetasr. According to r / c = K(n - r ) where r = moles of amino acid bound per mole of enzyme, c = moles of free amino acid, n = number of independent binding sites and K = association constant
12
t
=' 10 E
. 0
g 8 c
c
0
E
4
6
6 ' :
I
I
2
2
0
n
n
"2
6
10 14 18 Fraction numbers
22
26
.
Fig. 2. Isolation of the tyrosyl-tRNA synthetasr- tyrosylaclenylate complex by gel filtration. In the above experiment enzyme and substrates were incubated for 2.5 min at 18 "C in a total volume of 0.26 ml. Final concentrations were 0.35 mM [14C]ATP,0.39 mM ~-[~H]tyrosine and 54 pM enzyme. I n a parallel experiment 0.3 mM [Y-~'P]ATPwas used instead of [14C]ATP.(0)Enzyme from amino acid analysis (fractions 7 - 9) and absorbance at 280 nm (1 mg per ml equals 1.3 A-units per cm); (A) AMP and ATP, respectively; (0)L-tyrosine; (0)
496
Aminoacyl-tRNA Synthetases from Bacillus stearotherrnophilus
was really attributable to AMP in the tyrosyladenylate and not to a complex of ATP with enzyme, [ Y - ~ ~ P ] ATP was used in one experiment. All 32P-radioactivity eluted with the free ATP (Fig. 2). The possibility that tyrosyl-tRNA synthetase con0.0 ** tains two potential and equivalent active sites but IIP ; 0.7 \ **. that only one at a time may be occupied, i.e. that the second site can form adenylate only after the first 0.6 site has dissociated from its adenylate, was tested as 0.5 follows. Tyrosyladenylate-enzyme complex was prepared in the usual manner, isolated by gel filtration and incubated at a concentration of 5 pM with 0 2 4 6 8 10 12 200 pM ATP and 228 pM ~ - [ ~ H ] t y r o s i nfor e 2 min [ tRNAl( P.M ) at 18 "C and rechromatographed immediately. Of the Fig. 3. Fluorescence titrution of tyrosyl-tRNA synthetaw (2.7 p M ) recovered tyrosyladenylate-enzyme complex, 25 % had with tRNAs. (0) tRNATy' (1.2 nmol tyrosine acceptance per A,,, ~ - [ ~ H ] t y r o s i nincorporated. e This corresponds to a unit); (0) partly enriched tRNALeU(0.4 nmol leucine acceptance per A,,, unit); 0.1 M cacodylate pH 6.5, 10 mM MgCI,, 25 'C 15 % net incorporation of labelled tyrosine after correcting for the free enzyme formed by decomposition of the unlabelled complex for the duration of the experiment. In other words, the rate of formation of adenylate is reduced to the order of lop4 to s-l whereas the rate of de novo synthesis catalysed by the free enzyme is 18 s-' (A. R. Fersht, personal com125 150 munication).
\.
1
The Enzyme-tRNA Complex The fluorescence of tyrosyl-tRNA synthetase is reduced by 50 "/, upon saturation with tRNATyrfrom B . stearothermophilus (Fig. 3). No quenching was observed in the presence of a mixture of tRNA partially enriched in tRNAL2"but devoid of tyrosine acceptance activity. Analysis of the titration curve in Fig.3 by extrapolating the initial slope to the extrapolated terminal base line at high tRNA concentration [15] gives a stoichiometry of 1.9 tRNA binding sites per enzyme molecule. Collection of data at lower enzyme concentration revealed that the two sites are not equivalent (Fig. 4). The degree of fluorescence quenching is approximately 25 % for each site. The dissociation constants could be estimated (but not accurately determined, see [ 161) from experiments performed at enzyme concentrations below the first Kd and in between the two K,-values. The values at pH 6.5 (0.1 M cacodylate), 25 "C and in the presence of 10 mM MgCl, are 2 x lo-' M or less for the stronger site and around 1.3 x lop6 M for the weaker. Decreasing the pH to 5.4 (0.1 M sodium acetate) did not change the Kd-values within the limit of error which is, however, rather broad. But at pH 7.8 (0.144 M Tris/HCl) binding to both sites is weaker with Kdvalues of 0.7 x M and 5 x l o p 6 M. tRNA binding is independent of magnesium ions and Kd-values are unchanged between 0 and 1 5 m M MgCl, but decrease by a factor of 2 to 3 at 50 mM MgC1,.
.
"0
10
* .*:
x) Fo-F
.... 30
40
Fig.4. Scatchard plot f o r the binding of' tRNA' y' to tyrosyl-tRNA synrherase. F, is the fluorescence (arbitrary units) in the absence, and F in the presence of tRNATyr;enzyme (0.15 pM) in a 0.1 M cacodylate pH 6.5, 10 mM MgCI,, 25 "C. The limiting slopes drawn correspond to dissociation constants, K d ,of 0.14 and 1.25 x lo-' M. These values were determined in parallel experiments at 0.05 and 0.4 pM enzyme concentrations, respectively
Binding of tRNATyrfrom E. coli to the B. stearothermophilus enzyme exhibits the same two nonequivalent sites with similar affinities to those observed with the homologous system (data not shown).
DISCUSSION Before considering the implications of the results obtained the reliability of the data needs assessment, since binding studies on many similar enzymes have Eur. J. Biochem. 53 (1975)
14
H. R. Bosshard, G. L. E. Koch, and B. S. Hartley
been bedevilled by artefact. One of the main sources of error is inaccurate estimates of the concentration of active enzyme. We have determined the amount of enzyme protein in samples by direct amino acid analysis of hydrolysates and are confident that the error is 5%. However, this does not provide an estimate of the proportion of active species in the preparation. Since the preparation of enzyme used in this study was homogeneous by disc-gel electrophoresis, had the highest specific activity of seven independent preparations and crystallised easily, we have no reason to believe it contained significant amounts of inactive material. The binding of the small ligand tyrosine was measured by equilibrium dialysis and artefacts due to the Donnan effect or by adsorption to the membrane were not significant in our experiments. Stoichiometries were obtained from studies at high enzyme concentrations to enhance the reliability of the estimates. The binding of tRNA to the enzyme could not be measured by dialysis and fluorescence quenching had to be used for this purpose. The main problem with this technique is the ultraviolet absorption of the nucleic acid which must be corrected for. The formula derived by Ehrenberg et al. [14] was used for this correction since the total absorbances of enzyme and tRNA were within the prescribed limits. The other complication arose from the presence of more than one type of binding site for tRNA on the enzyme. For this reason estimates of the two dissociation constants could only be approximated. Use of gel filtration for studying the binding of aminoacyladenylate has been criticised on the grounds that the complex is not in equilibrium with the substrates throughout the isolation and would therefore yield under-estimates of the true stoichiometry. We have measured the rate of decomposition of the isolated enzyme-adenylate complex under the conditions of the isolation procedure and found it to have a half-life of over 20 h. Consequently it is unlikely that this factor affects the results of the experiment which lasts only about 20 min. Saturation of the available adenylate binding sites was evident from the independence of the measured stoichiometries on the enzyme and substrate concentrations used. The main source of error we have encountered was the high affinity of the enzyme for pyrophosphate. In the absence of pyrophosphatase only about 0.5 mol of aminoacyladenylate could be detected per mol of enzyme whereas this was doubled upon addition of pyrophosphatase. Further additions of pyrophosphatase did not increase the stoichiometry indicating that this source of error had been fully eliminated. Hence it is unlikely that the binding stoichiometries of tyrosine, tRNA and tyrosyladanylate to tyrosylEur. J. Biochem. 53 (1975)
497
tRNA synthetase are grossly incorrect. It is therefore significant that all these ligands manifest some asymmetry in their binding to the dimeric enzyme. Tyrosine and tyrosyladenylate bind to only one site on the enzyme under the conditions used. tRNATy‘has two binding sites on the enzyme but the affinities differ by almost one order of magnitude. This general asymmetry is somewhat surprising in view of the high degree of symmetry of the enzyme molecule in the crystal [ 2 ] . Assuming that this symmetry holds for the dimer in solution, one must conclude either that there is steric overlap between two adjacent binding sites or that substrate binding induces conformational asymmetry. There is support for the latter hypothesis from studies of the reaction of the native enzyme with 5,5’-dithio-bis-(2-nitrobenzoicacid) [l]. Of the four cysteine residues in the dimer only one reacted with the reagent and reaction was slow. Steric hindrance between two adjacent but relatively inaccessible thiol groups in a rigid dimer is again a possibility but stretches coincidence. An alternative assumption is of a major symmetrical conformation lacking ligand binding sites in equilibrium with a minor asymmetric form with a single ligand binding site. The thiol reactivity would be explained if the symmetrical form had four “buried” thiol groups and the asymmetric form had three “buried” and one “free” thiol groups. A similar explanation was adduced to explain the change in thiol reactivity of methionyl-tRNA synthetase of E. coli consequent on aminoacyladenylate formation [13]. Evidence is beginning to accumulate for functional and structural asymmetry in many synthetases and the point has been reached where some explanation for this phenomenon will have to be sought. One possibility is that the flip-flop type mechanism proposed by Harada and Wolfe [17] and Lazdunski et al. [18] operates in such cases. We have attempted to test this by examining the incorporation of [3H]tyrosine and ATP into the isolated enzyme-adenylate complex. If a flip-flop type process based on two equivalent active sites is operative we would expect the radioactivity to be incorporated into the complex at a rate equivalent to de n o w synthesis. The results obtained show that although there is some incorporation of radioactivity the rate is much slower than that observed for de novo synthesis. Thus a simple flip-flop mechanism is unlikely. Although two equivalent tyrosine and adenylate binding sites (or active sites, for the sake of argument) must be excluded from the available data, there is still the possibility that a second weak site escaped detection by our experimental approach. There is independent kinetic evidence for such a weak site
498
H. R. Bosshard, G. L. E. Koch, and B. S. Hartley: Aminoacyl-tRNA Synthetases from Bacillus stearoihermophilus
which forms but never accumulates tyrosyladenylate since its rate of hydrolysis (or dissociation) is faster than its rate of formation (A. Fersht and coworkers, unpublished). The existence of such a second weak site would, of course, be consistent with the asymmetric model of the functional enzyme deduced from our data. It will be most important to know whether this model has any significance for the catalytic specificity of tyrosyl-tRNA synthetase. H.R.B. is the recipient of a fellowship from the Schweizerischer Naiionaljonds.
REFERENCES 1. Koch, G . L. E. (1974) Biochemistry, 13, 2307-2312. 2. Reid, B. R., Koch, G. L. E., Boulanger, Y., Hartley, B. S. & Blow, D. M. (1973) J . Mol. Biol. 80, 199-201. 3. Hartley, B. S. (1974) Symp. SOC.Gen. Microbiol. 24, 151-182. 4. Loftfield, R. B. (1972) Progr. Nucfeic Acid Res. Mol. Biol. 12, 87- 128. 5. Knowles, J . R., Katze, J. R., Konigsberg, W. & SOH, D. (1970) J . Biol. Chem. 245, 1407- 1415.
6. Boecker, E. A. & Cantoni, G. L. (1973) Biochemisir.r., 12, 2384-2389. 7. Krajewska-Grynkiewicz, K., Buonocore, V. & Schlesinger, S. (1973) Biochim. Biophys. Acta, 312, 518-527. 8. Chousterman, S. & Chapeville, F. (1973) Eur. J . Biochem. 35, 51-56. 9. Levitzki, A., Stallcup, W. & Koshland, D. E., Jr (1971) Biochemistry, 10, 3371 - 3378. 10. Littauer, U. Z . , Yanofsky, S. A., Novogrodsky, A., Bursztyn, H., Galanter, Y . & Katchalski, E. (1969) Biochim. Biophys. Acta, 195, 29 - 49. 11. Pearson, R. L., Weiss, J. F. & Kelmers, A . D. (1971) Biochim. Biophys. Acta, 228, 770- 774. 12. Randerath, K. & Randerath, E. (1964) J . Chromatogr. 16, 111 - 125. 13. Bruton, C. J. & Hartley, B. S. (1970) J . Mol. Biol. 52, 165- 178. 14. Ehrenberg, M., Cronvall, E. & Rigler, R. (1971) FEBSLett. 18, 199- 203. 15. Velick, S. F., Parker, C. W. & Eisen, H. N. (1960) Proc. Nut1 Acad. Sci. U . S . A . 46, 1470-1482. 16. Weder, H. G., Schildknecht, T., Lutz, R. A. & Kesselring, P. (1974) Eur. J . Biochem. 42, 475-481. 17. Harada, K. & Wolfe, R. G . (1968) J . Bio/. C/zem. 243, 41314137. 18. Lazdunski, C., Petitclerc, C., Chappelet, D. & Lazdunski, C. (1971) Eur. J . Biochem. 20, 124- 139.
H. R. Bosshard and G . L. E. Koch, M.R.C. Laboratory of Molecular Biology, Postgraduate Medical School, University of Cambridge, Hills Road, Cambridge, Great Britain, CB2 2QH B. S. Hartley, Department of Biochemistry, Imperial College of Science and Technology, Prince-Consort Road, London, Great Britain, SW7 2AY
Eur. J. Biochem. 53 (1975)
