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Chloride affects the interaction between tyrosyl-tRNA synthetase and tRNA
Biochimica et Biophysica Acta 1472 (1999) 51^61 www.elsevier.com/locate/bba
Chloride a¡ects the interaction between tyrosyl-tRNA synthetase and tRNA R. Kalervo Airas * Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland Received 14 April 1999; received in revised form 1 June 1999; accepted 10 June 1999
Abstract The physiological concentration of free magnesium in Escherichia coli cells is about 1 mM, and there is almost no chloride in the cell. When the aminoacylation of tRNA by tyrosyl-tRNA synthetase was assayed at 1 mM free Mg2 , chloride (and sulphate) ions inhibited the reaction but acetate at the same concentration ( 6 200 mM) was not inhibitory. When the magnesium concentration was increased to 10 mM there was almost no chloride inhibition any more. Chloride strengthened 2 the PPi inhibition, the Kapp were 140, 120, and 56 WM at 0, 50 and 150 mM KCl, respectively. i (PPi ) values at 1 mM free Mg Chloride weakened the AMP inhibition, the corresponding values for Kapp i (AMP) were 0.35, 0.5, and 0.9 mM. The value of Tyr ) was clearly increased by chloride, being 22, 37, 93, and 240 nM at 0, 50, 100, and 150 mM KCl, respectively. Kapp m (tRNA Best-fit analyses of the PPi inhibition, AMP inhibition and Kapp m (tRNA) assays were accomplished using total rate equations. The analysis showed that the only kinetic events which are obligatory to explain the chloride effects are a weakened binding of Mg2 to the tRNA before the transfer reaction and a weakened binding of Mg2 to the Tyr-tRNAWenzyme complex after the transfer reaction. The dissociation constants for the former were 0.11, 0.3, and 2.8 mM and for the latter 0.6, 2.5, and 13 mM at 0, 50 and 150 mM KCl, respectively. Mg2 is required for the reactive conformation of tRNA in the transfer reaction but chloride weakens its formation. After the transfer reaction the dissociation of Mg2 from the aa-tRNAWenzyme complex enhances the dissociation of the aa-tRNA from the enzyme. The kinetics and the chloride effect were similar in the tyrosyl-tRNA synthetases from both Bacillus stearothermophilus and E. coli. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Synthetase; Tyrosyl-tRNA synthetase; Transfer ribonucleic acid; Chloride
1. Introduction Protein-nucleic acid interactions are, generally, sensitive to salt concentrations [1,2], but in some cases especially dramatic changes are found when the anionic part of the salt is changed without changing the salt concentration. Such examples have been listed by Ha et al. [3], and they include e.g. the lac
* Corresponding author.
repressor binding to DNA [3], the interaction of Escherichia coli RNA polymerase with the VPR promoter [2,4], and the interactions of some restriction enzymes with DNA [2]. In these cases the binding e¤ciency is increased 10^100 times when K-acetate or K-glutamate is substituted for KCl. Already in 1967 Loft¢eld and Eigner [5] analysed salt e¡ects on the valyl-tRNA synthetase from E. coli by the Debye^Hu«ckel theory. KCl, NaCl and K2 SO4 at relatively low concentrations of 33^133 mM strongly inhibited the aminoacylation. With the iso-
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 1 0 3 - 8
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leucyl-tRNA synthetase from E. coli Loft¢eld et al. [6] determined that the Kapp m (tRNA) value was increased 70 times, from 6 nM to 430 nM, when the NaCl concentration was increased from 0 to 100 mM. Their explanation for this strong e¡ect was by the so-called Hofmeister anion e¡ect on the water surfaces of the macromolecules. With many aminoacyl-tRNA synthetases Smith [7] found that not only the rate of aminoacylation but also the acceptance of the amino acids by tRNA was a¡ected by salts, typically in concentrations of 0.2^1 M. Yarus [8] measured the salt and solvent inhibition of isoleucyltRNA synthetase and found that also ammonium acetate was inhibitory at 1 M concentration but not at 0.5 M. The present work describes a chloride inhibition and its reversal by magnesium on tyrosyl-tRNA synthetases (TyrRS) from E. coli and Bacillus stearothermophilus, and that acetate is not inhibitory. The best¢t analyses of the PPi inhibitions and AMP inhibitions suggest that chloride remarkably weakens the binding of Mg2 to the tRNA and thereby the interaction between the enzyme and the tRNA. The concentration of free Mg2 in the E. coli cells has been reported to be as low as about 1 mM [9,10]. This is at a level where the chloride e¡ect is strong. Cl3 has been expressed to be `virtually absent' from
the E. coli cells [9]. The osmotic equilibrium of the cells is adjusted by K and organic anions, mainly glutamate [30]. 2. Materials and methods 2.1. Materials The highly puri¢ed preparation of TyrRS from E. coli C6 (E.c.TyrRS) was a gift from Dr. H. Sternbach, Go«ttingen. B.s.TyrRS was puri¢ed from B. stearothermophilus ATCC 12980 with a procedure which contained cell lysis with lysozyme, precipitation of nucleic acids with polyethylenimine, chromatography on DEAESepharose CL-6B at pH 6 using a gradient of 0^300 mM NaCl, solubilization of the ammonium sulphate-precipitated protein in a column of Sepharose CL-2B by a decreasing gradient of ammonium sulphate, gel ¢ltration on Sephacryl S-300, and chromatography on Poros Q ion exchanger at pH 6 using a gradient of 0^200 mM NaCl. The speci¢c activity of the preparation was 1.8 Wmol min31 mg31 . Unfractionated tRNA from E. coli MRE 600 (Boehringer) was used for both E.c.TyrRS and B.s.TyrRS.
Scheme 1. Mechanism of the tyrosyl-tRNA synthetase. aa (amino acid) is tyrosine, and it is bound before ATP. A conformational change EC*E is written between the activation and transfer steps. The role of Mg2 is not included in the scheme.
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2.2. Enzyme assays The rates of the aminoacylation of tRNA and ATP/PPi exchange reactions were assayed as described previously [11] except that chloride was replaced by acetate. The standard reaction mixture (100 Wl) for the aminoacylation contained 50 mM HEPES/25 mM KOH (pH 7.4 at 30³C), 0.02% chicken egg albumin, 1 mg/ml of tRNA, 2 mM ATP, about 50 000 cpm of 14 C-amino acid (0.7 WM), 5 WM non-radioactive amino acid, 3 mM Mg(acetate)2 , 100 mM K-acetate, and 1 mM dithiothreitol. Reaction temperature was 30³C. The ATP/PPi exchange activities were measured in a similar reaction mixture as the aminoacylation, but PPi (200 000 cpm, 2^15 WM) was substituted for the radioactive amino acid and 50 WM non-radioactive PPi was added.
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conformational change has been omitted are given in [12]. Eq. 1 gives the rate of the ATP/PPi exchange (in the presence of tRNA and AMP): V C21UC32=C23U C43 C41= C34 C41=C23=fD1UfC21=C12U C32=C23U C43=C34 C41=C34 C41=C23 C41=C12g D2UfC32=C23U C43 C41=C34 C41=C23g D3U C43 C41=C34 D4g:
1
Eq. 2 gives the rate of the aminoacylation of tRNA: V C41=fD1UfC21=C12U
2.3. Equations
C32=C23U C43=C34 C41=C34 C41=C23
The principle of derivation of the rate equations has been described previously [12,13]. The best-¢t mechanism for the E.c.TyrRS is given by Scheme 1, and the rate equations for that mechanism are in Eqs. 1 and 2. Scheme 2 shows the division of the total reaction into segments and the meaning of the C and D terms used in the equations (the term `segment' comes from Cha [14]). Eq. 3 lists the expressions of the C and D terms. When various mechanisms are tested, the C and D terms are rewritten. A change in the mechanism normally causes changes in only two or three of these terms, the derivation of the whole rate equation is not necessary. Therefore the grouping of the constants as in Scheme 2 serves as a simpli¢cation in the handling of the equations. The rate equations for the mechanisms where the
C41=C12g D2UfC32=C23U C43 C41=C34 C41=C23g D3U C43 C41=C34 D4g;
2
QPP PPM=K4M PPMM=K4MM PPF=K4; C12 K3USU=K2UAM=K1M; C21 G3U PPM=K4MU 1 R=K5 RR=K5M; C23 K6CU R=K5 RR=K5MU 1 QPP; C32 G6CU R=K5 RR=K5MU 1 QPP; C34 K6URR=K5M;
Scheme 2. Grouping of the reaction steps of Scheme 1 in four segments for the derivation of kinetic equations. The terms Cij [Ei ] express the rates between the segments and Di [Ei ] is the sum of the concentrations of the intermediates within the given segment. E1 is free E, E2 is EWTyr-AMP, E3 is *EWTyr-AMP, and E4 is *EWTyr-tRNA.
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C43 G6U M=KME4UAMP=K7; C41 K8 K8AMPUAMP=K7 K8AU AM=KAU 1 FM8UM=KME4; D1 1 SU=K2U 1 AM=K1M AF=K1 AMP=K7; D2 1 R=K5 RR=K5MU 1 QPP; D3 R=K5 RR=K5MU 1 QPP; D4 1 M=KME4U 1 AM=KA AMP=K7: 3 In the list M is the free Mg2 concentration. R0 is the total concentration of tRNA, RF the free tRNA and RR the `reactive tRNA' (MgtRNA). A0, AF, and AM mean total ATP, free ATP and MgATP concentrations, respectively, and PPF, PPM and PPMM mean free PPi , MgPPi and Mg2 PPi concentrations. The values of these ligand concentrations were calculated using their Kd values as described in [13]. SU means tyrosine. FM8 in term C41 expresses the Mg2 dependence of the k8X reaction: for instance the value of k8A in the presence of bound Mg2 is FM8Uk8A .
the ¢nal optimization of the constants the sum of the error percentages in the three di¡erent experiments was minimized. The term `global analysis' has also been used for this kind of procedure [16]. When calculated as above, the error percentages remain the same if all the rate constants in the equation are multiplied by the same number. This can be used to adjust the calculated kcat values to the same level as the measured kcat values. It also means that errors in the measured speci¢c activities do not change the reaction mechanism nor the relative rates in the di¡erent steps of the total reaction obtained in the optimization. The expressed rate constants give kcat values of the same order of magnitude as the measured kcat values. The standard deviations of the individual constants were estimated by the grid search method
2.4. Best-¢t analysis The analysis was based on the least-squares best-¢t analysis between the measured and calculated rate values [15]. The computer program for the analysis contained the following steps. A set of v values was calculated using Eqs. 1 and 2 to correspond to a set of measured v values from an experiment. The calculated values were adjusted to the same level as the measured values by setting the means equal. The root-mean-square values were calculated for the deviations between the measured and calculated values, and they were expressed as percentages of the mean. These error percentages are the measure of goodness of the ¢t. Original v values were used in the analysis although the 1/v values are expressed in Figs. 2 and 3. The kinetic constants were systematically varied, the error values were calculated, and those constant values were chosen which gave the lowest errors. In
Fig. 1. (A) E¡ect of potassium chloride and (B) e¡ect of TrisHCl bu¡er pH 7.4 on the rate of aminoacylation of tRNA by TyrRS. 100 mM K-acetate was always present. In A the bu¡er was 50 mM HEPES-KOH pH 7.4.
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Fig. 2. Dixon plots of the PPi inhibition of TyrRS when [Mg2 ] and [KCl] were varied. The plots were calculated using Eqs. 1 and 3 and the constant values listed in the experimental section and in Table 1. The error percentages expressing the goodness of ¢t were 4.1% in A, 3.0% in B and 5.8% in C.
[15]. The error limits are given only for the constants which are involved in the subject of the present work. Two limitations to the deviation values should be mentioned: the best-¢t value of one constant can be a¡ected by the values of some other constants, and when the mechanism is changed (by any change in Eq. 3) the best-¢t value can also be changed. In practice, twofold changes in some best-¢t values are possible when the mechanism is changed, but these changes become smaller when the model becomes closer to the real mechanism. (The same limitation applies generally in the calculations of kinetic con-
stants: the background assumptions must be correct for correct values.) The best-¢t values for the constants in the above mechanism were as follows: K1 = K1 = 400 WM (for free ATP); K1M = K1M = 640 þ 60 WM (for MgW ATP); KA = KA = 900 þ 250 WM (for MgWATP in segment 4); K2 = K2 = 19 þ 2 WM (for Tyr); G3 = k33 = 140 þ 20 s31 ; K4 = K4 = 200 WM (for free PPi ); K4M = K4M = 115 þ 15 WM (for MgWPPi ); K4MM = K4MM = 30 þ 10 WM (for Mg2 PPi ); K6 = k6 = 26 þ 12 s31 ; G6 = k36 = 18 þ 5 s31 ; K6C = k6C = 32 þ 8 s31 ; G6C = k36C = 6 þ 3 s31 ; K7 =
Fig. 3. AMP inhibition of the TyrRS reaction. The plots were obtained by best-¢t analysis as in Fig. 1, and the error percentages were 4.9% in A, 3.9% in B, and 4.5% in C.
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K7 = 100 WM; K8 = k8 = 7 þ 2.5 s31 ; K8A = k8A = 14 þ 4 s31 ; K8AMP = k8AMP = 1.6 þ 0.5 s31 ; F8 = 0.4. The value of k3 = 40 s31 was kept constant throughout the analysis, the value comes from [17]. 3. Results and discussion 3.1. Chloride inhibits the aminoacylation reaction Fig. 1 shows the e¡ect of KCl and Tris-HCl bu¡er on tyrosyl-tRNA synthesis when the magnesium concentration is varied. The physiological free [Mg2 ] of 1 mM is marked with a dashed line (as in other ¢gures). The chloride inhibition is observed below about 3 mM free [Mg2 ] but above this value no big changes in the activities are found. The e¡ect of 150 mM Tris bu¡er (125 mM Cl3 ) is still somewhat stronger than of 50 mM Tris bu¡er+100 mM KCl. Potassium acetate (100 mM) was present to keep the [K ] high. Sulphate also strongly inhibited. 50 mM K2 SO4 caused the same e¡ect as 150 mM KCl. Potassium acetate did not cause inhibition (below 200 mM), although it lies between sulphate and chloride in the Hofmeister anion series [18]. HEPES anion (25^ 125 mM) in the HEPES-KOH bu¡er was also without inhibiting e¡ect. The salt e¡ect is a sum of di¡erent e¡ects such as activation by potassium ions at concentrations below 100 mM and inhibition by chloride (or sulphate) at low Mg2 concentrations. If 100 mM K-acetate was added to keep the K concentration high, even low chloride concentrations ( 6 50 mM) were inhibitory, but without K-acetate, 50 mM KCl slightly activated when the K activation was stronger than the Cl3 inhibition. The apparent Michaelis constants for tRNATyr were strongly increased by KCl. The Hanes plots were essentially straight lines (at 1 mM free Mg2 , 2 mM MgATP, 1 WM Tyr). The Kapp m (tRNA) values were 22, 37, 93 and 240 nM at 0, 50, 100, and 150 mM KCl, respectively. 3.2. Chloride e¡ects on the PPi inhibition Chloride strengthened the PPi inhibition at all [Mg2 ] values (Fig. 2), although the apparent chlo-
ride inhibition was observed at low magnesium concentrations. The Kapp i (PPi ) values at 1 mM free magnesium were 140, 120, and 56 WM at 0, 50, and 150 mM KCl, respectively. 50 mM K2 SO4 caused almost exactly the same e¡ect as 150 mM KCl in Fig. 2C. In general, the PPi inhibition is strengthened if the dissociation constant for PPi is lowered, or if the pyrophosphorolysis rate k33 is increased, or if the rate forwards in the next steps are lowered. 3.3. Chloride e¡ects on the AMP inhibition AMP inhibits the aminoacylation typically with values of about 0.3^0.7 mM. Chloride had almost no e¡ect on the AMP inhibition at high (8 mM free) magnesium concentrations but at low (below 1 mM free) magnesium concentrations the inhibition was clearly weakened (Fig. 3). The Kapp i (AMP) values at 1 mM free magnesium were 0.35, 0.5, and 0.9 mM at 0, 50, and 150 mM KCl, respectively. The AMP inhibition is weakened if the dissociation constant (K7 ) of AMP is increased or if the reverse transfer reaction (k36 ) is lowered or if the rate of the dissociation of the aminoacyl-tRNA from the enzyme is increased. Weaker e¡ects on the inhibition may come from other parts of the total reaction. The parallel shift upwards of the plot at 1.5 mM Mg2 with increasing KCl concentrations can be simulated by increasing the value of KME4 . (The parallel shift is stronger in B.s.TyrRS than in E.c.TyrRS.) Kapp i
3.4. Some details of the mechanism Fig. 4 shows some kinetic experiments used in designing the detailed mechanism for the best-¢t analysis. The KTyr ( = K2 ) value from the intersection point in Fig. 4A is 19 WM. In Fig. 4B a sharp peak at about 0.5 mM Mg2 free is observed. The fall in the ATP/PPi exchange rate above this magnesium concentration can be almost totally explained by the decrease in the [MgPPi ] by formation of Mg2 PPi . The reactive species is MgPPi (e.g. due to the microscopic reversibility when MgATP is the reactive compound in the forward reaction) and the Kd value for the dissociation of one magnesium from Mg2 PPi is as low as 2.6 mM [19]. In the absence of tRNA an additional inhibiting binding of magnesium in seg-
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Fig. 4. Some kinetic experiments on TyrRS from E. coli. (A) Hanes plots of the aminoacylation reaction when [Tyr] and [MgATP] were varied. (B) Dependence of the ATP/PPi exchange rate on magnesium, AMP, and tRNA concentrations. (C) PPi dependence of the relation of the rates of the ATP/PPi exchange and aminoacylation of tRNA at identical conditions. [Mg2 ]total was 3 mM+[PPi ]. (D) Hanes plots of the ATP dependence of the aminoacylation reaction and inhibition by AMP. The concentration of Mg(acetate)2 was 10 mM+[ATP].
ment 2 improves the ¢t. In the presence of tRNA this inhibiting binding of magnesium is very weak and in the presence of chloride not observed. Therefore it was not taken into account in Eq. 3 nor in the analyses of Figs. 2 and 3. In Fig. 4B the exchange rate is reduced in the presence of tRNA because the enzyme is mainly in the late intermediates (segments 3 and 4 in Scheme 2) and thus not available in the exchange reaction. In Fig. 4C the relation vexchange /vacylation was measured. The kinetic equations for the relation are very
simple: if the conformational change (k6C ) is not included vexch /vacyl = C21 /C24 [12], and if it is included, vexch /vacyl = (C21 /C23 )U(C34 +C32 )/C34 . (The terms are from Scheme 2.) The expressions contain only a limited amount of rate and equilibrium constants and therefore it is useful in an estimation of the detailed mechanism. Straight lines in Fig. 4C were expected. The clearly curved plots were ¢tted only if both PPi and tRNA could be bound to the enzyme during the conformational-change step. The value of vexch /vacyl Tyr (at 1 mM Mg2 , 100 WM PPi ) was free , 1 WM tRNA
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about 1 in Fig. 4C but at optimal conditions the rate of the ATP/PPi exchange (without tRNA) was 8.7 times higher than the aminoacylation rate (without PPi ). The e¡ect of AMP on the Hanes plots for the ATP dependence of the aminoacylation (Fig. 4D) can be used when the events after the transfer reaction are analysed. The intersection on the ordinate axis would mean an uncompetitive inhibition mechanism (from segment 4). A competitive inhibition (from segment 1) would give parallel lines. Curved plots suggest a second, activating binding of ATP. Fig. 4D shows that the AMP inhibition almost totally comes from segment 4.
b
b
b
3.5. Best-¢t analysis The principle of `global analysis' [17] was applied, i.e. the same detailed reaction mechanism and the same constant values were used for several di¡erent experiments. The main purpose was to ¢nd the correct mechanism, which includes testing the possible steps, the relative rates of the steps and which ligands are bound at a given step and in which order. If the mechanism is correct, the error rates in the analyses of all di¡erent experiments should be low. If the error of the assay (sxWy of the rate curve) is 2^3%, errors of 3^5% are attained with a good mechanism but a weak mechanism gives errors of tens of per cent. Over 10% average errors were regarded as non-acceptable. Simultaneous analysis of several, chosen, experiments is an e¤cient way to reject wrong mechanisms and to ¢nd the steps of the complex reaction in which the observed features are connected. The same is not true if the best-¢t analysis is based on only one experiment where two ligand concentrations are varied. The following list gives the details of the mechanism and the reasons for their use: b
b
Only one Mg2 is assumed to be bound to tRNA to make it reactive in the transfer reaction. Several magnesium ions are known to be bound to tRNA [20], but in the simplest model the present system behaves as if only one Mg2 would be responsible for the structural change. The concentrations of di¡erent forms of ATP and PPi (free ATP, MgATP, free PPi , MgPPi , Mg2 PPi )
b
b
b
are calculated for every measurement point using the Kd values, and so they are taken into account in the analysis. The amino acid is bound to the enzyme before ATP [21]. Both the random model and the model where two ATP molecules are required were weaker in the best-¢t analysis, especially when measured by the ATP/PPi exchange. tRNA and ATP do not a¡ect the binding of each other. This is not necessarily true, probably tRNA and ATP weaken the binding of each other, but the assumption was made for simplicity since it has only a very weak e¡ect in the experiments of this work. AMP can be bound to the ATP site before the activation reaction. The e¡ect of this binding, however, is very weak (Fig. 4D), the stronger inhibition comes from the steps after the transfer reaction. A conformational change between the activation and transfer steps is included in the mechanism, as earlier [13]. It is a reorganization of the active site for the transfer reaction, and the change can happen in the enzyme, tRNA or both. Such changes are observed in the crystal structures when tRNA is bound [22^25] but the existence in the kinetic mechanism has not been self-evident. The present data, the chloride e¡ects, could not be satisfactorily ¢tted if this step was omitted. Some data (e.g. the curvature in Fig. 4C) required that the above conformational change can occur also when PPi is bound and also with both free tRNA and MgtRNA. In the transfer reaction PPi is not bound and MgtRNA is required. The dissociation of the aminoacyl-tRNA from the enzyme is enhanced by two events: dissociation of the magnesium ion from the enzymeWaa-tRNA complex [13] and binding of ATP to the complex (maybe to the AMP site) [12]. Both the experiments in Fig. 4D and the best ¢t analysis of the chloride e¡ect strongly support this model. Although the gross model shows the involvement of the two events, the details remain to be solved. A rate-limiting step at the end of the total aminoacylation reaction has often been detected with di¡erent aminoacyl-tRNA synthetases [26]. The slow step (k8 , k8A , k8AMP ) in the equations is thought to be a conformational change (maybe
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Table 1 Dependences of some kinetic constants on [KCl] [KCl] (mM)
KMR (mM)
KME4 (mM)
K5M (WM)
K5 (WM)
0 50 150
0.11 þ 0.05 0.3 þ 0.06 2.8 þ 0.8
0.6 þ 0.14 2.5 þ 1.2 V13
0.42 þ 0.12 0.5 þ 0.1 0.6 þ 0.14
1.2 1.4 4.5
KMR is Kd of Mg2 from MgtRNA, KME4 is Kd of Mg2 from the complex enzymeWTyr-tRNAWMg, K5M is Kd of MgtRNA from enzymeWMgtRNA, and K5 is Kd of tRNA from (non-reactive) enzymeWtRNA.
corresponding to that with k36C ) which precedes a rapid dissociation of the product.
The detailed kinetic mechanism of the chloride effect is not known and was not written into the equations. The di¡erent chloride concentrations were analysed separately. Three di¡erent experiments were included in the `global'-type analysis, and the sum of the error percentages was minimized when the constant values were optimized. The experiments were the PPi inhibition (Fig. 2), the AMP inhibition (Fig. 3), and the Kapp m (tRNA) assay. Very simple changes in the constant values are required to obtain an excellent ¢t (3^5% error) at di¡erent KCl concentrations (Table 1), and all these changes are involved in the binding or dissociation of tRNA from the enzyme. The minimal changes for a satisfactory ¢t (5^6% average error) would be the increases only in the KMR and KME4 values. (These two constants may represent the same event, dissociation of Mg2 from MgWtRNA, at di¡erent steps of the total reaction.) Table 1 also shows that in the best-¢t system there is almost no change with increasing [KCl] in the real dissociation constant, K5M , of the reactive species MgtRNA. The big change in the Kapp m (tRNA) value comes through the weakened binding of Mg2 to tRNA. 3.6. Comparison of the tyrosyl-tRNA synthetases from E. coli and B. stearothermophilus The kinetic mechanism of the TyrRS from B. stearothermophilus has been thoroughly studied previously [17,27^29]. When the constant values and mechanism of those works were applied in the best¢t analyses of the present work, not even a close ¢t was attained. The minimum errors were tens of per cents. Major changes both in the mechanism and in
values of some individual constants were required. Therefore some kinetic experiments were also done with the TyrRS from B. stearothermophilus (Fig. 5). The similarity is striking between the results in Fig. 5 and the corresponding or relating experiments with the E. coli enzyme in Figs. 1 and 4. The experiments show that there are no big di¡erences in the mechanisms of TyrRS:s from the two bacteria. The relation vexch /vacyl was lower in B.s.TyrRS than in E.c.TyrRS. However at optimal conditions the rate of the ATP/ PPi exchange (without tRNA) was 7.3 times higher than the aminoacylation rate (without PPi ), thus the relation is not essentially di¡erent from the value of 8.7 for the E.c.TyrRS. The details of the strong reduction of the ATP/PPi exchange rate of B.s.TyrRS by tRNA have not yet been analysed. The analysis of the KCl e¡ects on the PPi inhibition and AMP inhibition showed that the KCl e¡ect was somewhat stronger with the B. s. enzyme (with tRNA from E. 2 coli). The Kapp i (PPi ) values (at 1 mM [Mg ]free ) were app 230, 100, and 48 WM, and the Ki (AMP) values were 0.32, 0.50, and 2.15 mM at 0, 50, and 150 mM KCl, respectively. Some di¡erences existed in the measurement conditions between the previous studies with B. s. TyrRS and the present work: typically in the previous studies the chloride concentration was about 130 mM (100 mM from the bu¡er, 30 mM from MgCl2 ), [Mg2 ]free was 10 mM, and the pH of the Tris-HCl bu¡er was 7.78 [17,28], while in the present work no Cl3 , about 1 mM free Mg2 , and the HEPES-KOH bu¡er pH 7.4 were used. The di¡erences in the chloride and magnesium concentrations seem to be su¤cient to explain most of the observed di¡erences. If high chloride concentrations are used, high Mg2 concentrations must be used for optimal activity (Fig. 1). If high magnesium concentrations are also systematically used in the presence of PPi in the measurements of pyrophosphorolysis or ATP/PPi ex-
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Fig. 5. Some kinetic experiments on TyrRS from B. stearothermophilus. A is like Fig. 1A, D like Fig. 1B, B like Fig. 4A, C like Fig. 4B. E is like Fig. 4C but [Mg2 ] is varied instead of [tRNA]. F is like Fig. 4C but [Mg2 ]total is 1 mM+[ATP].
change, most of PPi is in the form of Mg2 PPi , while MgPPi would be the reactive compound. From the Kd value (2.6 mM), 78% of PPi is in the form of Mg2 PPi at 10 mM free Mg2 . The lowered concentration of the reactive species MgPPi should be taken into account in the calculations of the pyrophosphorolysis rates and kinetic constants through the ATP/ PPi exchange measurements, but in the works mentioned above it has not been taken into account. In the earlier studies [17] with B.s.TyrRS some constant values were k33 = 16.6 s31 , KPP = 0.61 mM, and KATP = 4.7 mM, but the corresponding values in the present work were 120 s31 , 0.23 mM and 0.45 mM. The di¡erences in k33 and KPP can be explained by the presence of Mg2 PPi , but the reason for the big di¡erence in the KATP value remains obscure. The low Kapp m (ATP) value from Fig. 5F (160 WM) would support the low KATP value. The analyses of the previous studies did not include a slow step after
the transfer reaction, but the present data could not be ¢tted without the slow step. 3.7. Nature of the chloride e¡ect The molecular basis of the lowered a¤nity between the enzyme and tRNA in the presence of chloride (or sulphate) is not known. The chloride e¡ect resembles the other cases of protein-nucleic acid interactions where replacement of chloride by acetate or glutamate strengthens the interaction [2^ 4]. For instance, the complex formation between RNA polymerase and the VPR promoter is a multistep process where chloride favours one conformation but Mg2 another, transcriptionally competent, conformation [4]. In an analogous way, chloride may favour a non-reactive conformation of TyrRS or the CCA end of tRNA, but binding of Mg2 causes the formation of the reactive conformation.
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It looks like the e¡ect in the present work would not be principally caused by salt concentration since K-acetate does not inhibit at the concentrations used. Hofmeister anion e¡ect on the water surface of the macromolecules is also not an obvious explanation. More speci¢c e¡ects of the anions may be necessary. Sulphate, acetate and chloride belong to the stabilizing end of the Hofmeister series [18] but of these sulphate is the strongest inhibitor and has the strongest e¡ect on the PPi inhibition while acetate has almost no e¡ect. 3.8. On the enzyme assays Most assays of the aminoacyl-tRNA synthetases have been done in Tris-HCl bu¡ers and potassium chloride has been added to increase the K concentration. The present results suggest that these conditions should be reconsidered. In normal assays of the aminoacylation activity the e¡ect of high chloride concentration is reversed at high Mg2 concentrations, and therefore is not serious (at least in TyrRS). However, when reaction mechanisms including binding of tRNA are studied, or if PPi is used in the measurements, the e¡ects of high chloride and magnesium concentrations should be taken into account. Furthermore, MgCl2 or MgSO4 should not be used in the studies of the Mg2 e¡ects since the interacting anion concentration is changed at the same time. Chloride can also have some e¡ects in the measurements of the proofreading since the correction systems are placed between the binding of the tRNA and dissociation of the aa-tRNA. References [1] M.T. Record Jr., W. Zhang, C.F. Anderson, Adv. Protein Chem. 51 (1998) 281^353. [2] S. Leirmo, C. Harrison, D.S. Cayley, R.R. Burgess, M.T. Record Jr., Biochemistry 26 (1987) 2095^2101. [3] J.-H. Ha, M.W. Capp, M.D. Hohenwalter, M. Baskerville, M.T. Record Jr., J. Mol. Biol. 228 (1992) 252^264.
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[4] W.C. Suh, S. Leirmo, M.T. Record Jr., Biochemistry 31 (1992) 7815^7825. [5] R.B. Loft¢eld, E.A. Eigner, J. Biol. Chem. 242 (1967) 5355^ 5359. [6] R.B. Loft¢eld, E.A. Eigner, A. Pastuszyn, T.N.E. Lo«vgren, H. Jakubowski, Proc. Natl. Acad. Sci. USA 77 (1980) 3374^ 3378. [7] D.W.E. Smith, J. Biol. Chem. 244 (1969) 896^901. [8] M. Yarus, Biochemistry 11 (1972) 2050^2060. [9] E. Kellenberger, in: F.C. Neidhardt (Ed.), Escherichia coli and Salmonella. Cellular and Molecular Biology, ASM Press, Washington, DC, 1996, pp. 17^28. [10] T. Alatossava, H. Ju«tte, A. Kuhn, E. Kellenberger, J. Bacteriol. 162 (1985) 413^419. [11] R.K. Airas, Eur. J. Biochem. 192 (1990) 401^409. [12] R.K. Airas, Eur. J. Biochem. 210 (1992) 443^450. [13] R.K. Airas, Eur. J. Biochem. 240 (1996) 223^231. [14] S. Cha, J. Biol. Chem. 243 (1968) 820^825. [15] M.L. Johnson, L.M. Faunt, Methods Enzymol. 210 (1992) 1^37. [16] J.M. Beechem, Methods Enzymol. 210 (1992) 37^54. [17] D.M. Lowe, G. Winter, A.R. Fersht, Biochemistry 26 (1987) 6038^6043. [18] M.G. Cacace, E.M. Landau, J.J. Ramsden, Q. Rev. Biophys. 30 (1997) 241^277. [19] S.E. Volk, A. Baykov, V.S. Duzhenko, S.M. Avaeva, Eur. J. Biochem. 125 (1982) 215^220. [20] D.C. Lynch, P.R. Schimmel, Biochemistry 13 (1974) 1841^ 1852. [21] P. Brick, T.N. Bhat, D.M. Blow, J. Mol. Biol. 208 (1988) 83^98. [22] S. Cusack, A. Yaremchuk, M. Tukalo, EMBO J. 15 (1996) 2834^2842. [23] S. Cusack, A. Yaremchuk, M. Tukalo, EMBO J. 15 (1996) 6321^6334. [24] B. Rees, J. Cavarelli, D. Moras, Biochimie 78 (1996) 624^ 631. [25] M.A. Rould, J.J. Perona, D. So«ll, T.A. Steitz, Science 246 (1989) 1135^1142. [26] P.R. Schimmel, D. So«ll, Annu. Rev. Biochem. 48 (1979) 601^648. [27] A.R. Fersht, J.W. Knill-Jones, H. Bedouelle, G. Winter, Biochemistry 27 (1988) 1581^1587. [28] T.N.C. Wells, J.W. Knill-Jones, T.E. Gray, A.R. Fersht, Biochemistry 30 (1991) 5151^5156. [29] J.M. Avis, A.G. Day, G.A. Garcia, A.R. Fersht, Biochemistry 32 (1993) 5312^5320. [30] E.A. Galinski, Adv. Microb. Physiol. 37 (1996) 273^328.
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Chloride a¡ects the interaction between tyrosyl-tRNA synthetase and tRNA R. Kalervo Airas * Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland Received 14 April 1999; received in revised form 1 June 1999; accepted 10 June 1999
Abstract The physiological concentration of free magnesium in Escherichia coli cells is about 1 mM, and there is almost no chloride in the cell. When the aminoacylation of tRNA by tyrosyl-tRNA synthetase was assayed at 1 mM free Mg2 , chloride (and sulphate) ions inhibited the reaction but acetate at the same concentration ( 6 200 mM) was not inhibitory. When the magnesium concentration was increased to 10 mM there was almost no chloride inhibition any more. Chloride strengthened 2 the PPi inhibition, the Kapp were 140, 120, and 56 WM at 0, 50 and 150 mM KCl, respectively. i (PPi ) values at 1 mM free Mg Chloride weakened the AMP inhibition, the corresponding values for Kapp i (AMP) were 0.35, 0.5, and 0.9 mM. The value of Tyr ) was clearly increased by chloride, being 22, 37, 93, and 240 nM at 0, 50, 100, and 150 mM KCl, respectively. Kapp m (tRNA Best-fit analyses of the PPi inhibition, AMP inhibition and Kapp m (tRNA) assays were accomplished using total rate equations. The analysis showed that the only kinetic events which are obligatory to explain the chloride effects are a weakened binding of Mg2 to the tRNA before the transfer reaction and a weakened binding of Mg2 to the Tyr-tRNAWenzyme complex after the transfer reaction. The dissociation constants for the former were 0.11, 0.3, and 2.8 mM and for the latter 0.6, 2.5, and 13 mM at 0, 50 and 150 mM KCl, respectively. Mg2 is required for the reactive conformation of tRNA in the transfer reaction but chloride weakens its formation. After the transfer reaction the dissociation of Mg2 from the aa-tRNAWenzyme complex enhances the dissociation of the aa-tRNA from the enzyme. The kinetics and the chloride effect were similar in the tyrosyl-tRNA synthetases from both Bacillus stearothermophilus and E. coli. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Synthetase; Tyrosyl-tRNA synthetase; Transfer ribonucleic acid; Chloride
1. Introduction Protein-nucleic acid interactions are, generally, sensitive to salt concentrations [1,2], but in some cases especially dramatic changes are found when the anionic part of the salt is changed without changing the salt concentration. Such examples have been listed by Ha et al. [3], and they include e.g. the lac
* Corresponding author.
repressor binding to DNA [3], the interaction of Escherichia coli RNA polymerase with the VPR promoter [2,4], and the interactions of some restriction enzymes with DNA [2]. In these cases the binding e¤ciency is increased 10^100 times when K-acetate or K-glutamate is substituted for KCl. Already in 1967 Loft¢eld and Eigner [5] analysed salt e¡ects on the valyl-tRNA synthetase from E. coli by the Debye^Hu«ckel theory. KCl, NaCl and K2 SO4 at relatively low concentrations of 33^133 mM strongly inhibited the aminoacylation. With the iso-
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 9 ) 0 0 1 0 3 - 8
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leucyl-tRNA synthetase from E. coli Loft¢eld et al. [6] determined that the Kapp m (tRNA) value was increased 70 times, from 6 nM to 430 nM, when the NaCl concentration was increased from 0 to 100 mM. Their explanation for this strong e¡ect was by the so-called Hofmeister anion e¡ect on the water surfaces of the macromolecules. With many aminoacyl-tRNA synthetases Smith [7] found that not only the rate of aminoacylation but also the acceptance of the amino acids by tRNA was a¡ected by salts, typically in concentrations of 0.2^1 M. Yarus [8] measured the salt and solvent inhibition of isoleucyltRNA synthetase and found that also ammonium acetate was inhibitory at 1 M concentration but not at 0.5 M. The present work describes a chloride inhibition and its reversal by magnesium on tyrosyl-tRNA synthetases (TyrRS) from E. coli and Bacillus stearothermophilus, and that acetate is not inhibitory. The best¢t analyses of the PPi inhibitions and AMP inhibitions suggest that chloride remarkably weakens the binding of Mg2 to the tRNA and thereby the interaction between the enzyme and the tRNA. The concentration of free Mg2 in the E. coli cells has been reported to be as low as about 1 mM [9,10]. This is at a level where the chloride e¡ect is strong. Cl3 has been expressed to be `virtually absent' from
the E. coli cells [9]. The osmotic equilibrium of the cells is adjusted by K and organic anions, mainly glutamate [30]. 2. Materials and methods 2.1. Materials The highly puri¢ed preparation of TyrRS from E. coli C6 (E.c.TyrRS) was a gift from Dr. H. Sternbach, Go«ttingen. B.s.TyrRS was puri¢ed from B. stearothermophilus ATCC 12980 with a procedure which contained cell lysis with lysozyme, precipitation of nucleic acids with polyethylenimine, chromatography on DEAESepharose CL-6B at pH 6 using a gradient of 0^300 mM NaCl, solubilization of the ammonium sulphate-precipitated protein in a column of Sepharose CL-2B by a decreasing gradient of ammonium sulphate, gel ¢ltration on Sephacryl S-300, and chromatography on Poros Q ion exchanger at pH 6 using a gradient of 0^200 mM NaCl. The speci¢c activity of the preparation was 1.8 Wmol min31 mg31 . Unfractionated tRNA from E. coli MRE 600 (Boehringer) was used for both E.c.TyrRS and B.s.TyrRS.
Scheme 1. Mechanism of the tyrosyl-tRNA synthetase. aa (amino acid) is tyrosine, and it is bound before ATP. A conformational change EC*E is written between the activation and transfer steps. The role of Mg2 is not included in the scheme.
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2.2. Enzyme assays The rates of the aminoacylation of tRNA and ATP/PPi exchange reactions were assayed as described previously [11] except that chloride was replaced by acetate. The standard reaction mixture (100 Wl) for the aminoacylation contained 50 mM HEPES/25 mM KOH (pH 7.4 at 30³C), 0.02% chicken egg albumin, 1 mg/ml of tRNA, 2 mM ATP, about 50 000 cpm of 14 C-amino acid (0.7 WM), 5 WM non-radioactive amino acid, 3 mM Mg(acetate)2 , 100 mM K-acetate, and 1 mM dithiothreitol. Reaction temperature was 30³C. The ATP/PPi exchange activities were measured in a similar reaction mixture as the aminoacylation, but PPi (200 000 cpm, 2^15 WM) was substituted for the radioactive amino acid and 50 WM non-radioactive PPi was added.
53
conformational change has been omitted are given in [12]. Eq. 1 gives the rate of the ATP/PPi exchange (in the presence of tRNA and AMP): V C21UC32=C23U C43 C41= C34 C41=C23=fD1UfC21=C12U C32=C23U C43=C34 C41=C34 C41=C23 C41=C12g D2UfC32=C23U C43 C41=C34 C41=C23g D3U C43 C41=C34 D4g:
1
Eq. 2 gives the rate of the aminoacylation of tRNA: V C41=fD1UfC21=C12U
2.3. Equations
C32=C23U C43=C34 C41=C34 C41=C23
The principle of derivation of the rate equations has been described previously [12,13]. The best-¢t mechanism for the E.c.TyrRS is given by Scheme 1, and the rate equations for that mechanism are in Eqs. 1 and 2. Scheme 2 shows the division of the total reaction into segments and the meaning of the C and D terms used in the equations (the term `segment' comes from Cha [14]). Eq. 3 lists the expressions of the C and D terms. When various mechanisms are tested, the C and D terms are rewritten. A change in the mechanism normally causes changes in only two or three of these terms, the derivation of the whole rate equation is not necessary. Therefore the grouping of the constants as in Scheme 2 serves as a simpli¢cation in the handling of the equations. The rate equations for the mechanisms where the
C41=C12g D2UfC32=C23U C43 C41=C34 C41=C23g D3U C43 C41=C34 D4g;
2
QPP PPM=K4M PPMM=K4MM PPF=K4; C12 K3USU=K2UAM=K1M; C21 G3U PPM=K4MU 1 R=K5 RR=K5M; C23 K6CU R=K5 RR=K5MU 1 QPP; C32 G6CU R=K5 RR=K5MU 1 QPP; C34 K6URR=K5M;
Scheme 2. Grouping of the reaction steps of Scheme 1 in four segments for the derivation of kinetic equations. The terms Cij [Ei ] express the rates between the segments and Di [Ei ] is the sum of the concentrations of the intermediates within the given segment. E1 is free E, E2 is EWTyr-AMP, E3 is *EWTyr-AMP, and E4 is *EWTyr-tRNA.
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C43 G6U M=KME4UAMP=K7; C41 K8 K8AMPUAMP=K7 K8AU AM=KAU 1 FM8UM=KME4; D1 1 SU=K2U 1 AM=K1M AF=K1 AMP=K7; D2 1 R=K5 RR=K5MU 1 QPP; D3 R=K5 RR=K5MU 1 QPP; D4 1 M=KME4U 1 AM=KA AMP=K7: 3 In the list M is the free Mg2 concentration. R0 is the total concentration of tRNA, RF the free tRNA and RR the `reactive tRNA' (MgtRNA). A0, AF, and AM mean total ATP, free ATP and MgATP concentrations, respectively, and PPF, PPM and PPMM mean free PPi , MgPPi and Mg2 PPi concentrations. The values of these ligand concentrations were calculated using their Kd values as described in [13]. SU means tyrosine. FM8 in term C41 expresses the Mg2 dependence of the k8X reaction: for instance the value of k8A in the presence of bound Mg2 is FM8Uk8A .
the ¢nal optimization of the constants the sum of the error percentages in the three di¡erent experiments was minimized. The term `global analysis' has also been used for this kind of procedure [16]. When calculated as above, the error percentages remain the same if all the rate constants in the equation are multiplied by the same number. This can be used to adjust the calculated kcat values to the same level as the measured kcat values. It also means that errors in the measured speci¢c activities do not change the reaction mechanism nor the relative rates in the di¡erent steps of the total reaction obtained in the optimization. The expressed rate constants give kcat values of the same order of magnitude as the measured kcat values. The standard deviations of the individual constants were estimated by the grid search method
2.4. Best-¢t analysis The analysis was based on the least-squares best-¢t analysis between the measured and calculated rate values [15]. The computer program for the analysis contained the following steps. A set of v values was calculated using Eqs. 1 and 2 to correspond to a set of measured v values from an experiment. The calculated values were adjusted to the same level as the measured values by setting the means equal. The root-mean-square values were calculated for the deviations between the measured and calculated values, and they were expressed as percentages of the mean. These error percentages are the measure of goodness of the ¢t. Original v values were used in the analysis although the 1/v values are expressed in Figs. 2 and 3. The kinetic constants were systematically varied, the error values were calculated, and those constant values were chosen which gave the lowest errors. In
Fig. 1. (A) E¡ect of potassium chloride and (B) e¡ect of TrisHCl bu¡er pH 7.4 on the rate of aminoacylation of tRNA by TyrRS. 100 mM K-acetate was always present. In A the bu¡er was 50 mM HEPES-KOH pH 7.4.
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Fig. 2. Dixon plots of the PPi inhibition of TyrRS when [Mg2 ] and [KCl] were varied. The plots were calculated using Eqs. 1 and 3 and the constant values listed in the experimental section and in Table 1. The error percentages expressing the goodness of ¢t were 4.1% in A, 3.0% in B and 5.8% in C.
[15]. The error limits are given only for the constants which are involved in the subject of the present work. Two limitations to the deviation values should be mentioned: the best-¢t value of one constant can be a¡ected by the values of some other constants, and when the mechanism is changed (by any change in Eq. 3) the best-¢t value can also be changed. In practice, twofold changes in some best-¢t values are possible when the mechanism is changed, but these changes become smaller when the model becomes closer to the real mechanism. (The same limitation applies generally in the calculations of kinetic con-
stants: the background assumptions must be correct for correct values.) The best-¢t values for the constants in the above mechanism were as follows: K1 = K1 = 400 WM (for free ATP); K1M = K1M = 640 þ 60 WM (for MgW ATP); KA = KA = 900 þ 250 WM (for MgWATP in segment 4); K2 = K2 = 19 þ 2 WM (for Tyr); G3 = k33 = 140 þ 20 s31 ; K4 = K4 = 200 WM (for free PPi ); K4M = K4M = 115 þ 15 WM (for MgWPPi ); K4MM = K4MM = 30 þ 10 WM (for Mg2 PPi ); K6 = k6 = 26 þ 12 s31 ; G6 = k36 = 18 þ 5 s31 ; K6C = k6C = 32 þ 8 s31 ; G6C = k36C = 6 þ 3 s31 ; K7 =
Fig. 3. AMP inhibition of the TyrRS reaction. The plots were obtained by best-¢t analysis as in Fig. 1, and the error percentages were 4.9% in A, 3.9% in B, and 4.5% in C.
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K7 = 100 WM; K8 = k8 = 7 þ 2.5 s31 ; K8A = k8A = 14 þ 4 s31 ; K8AMP = k8AMP = 1.6 þ 0.5 s31 ; F8 = 0.4. The value of k3 = 40 s31 was kept constant throughout the analysis, the value comes from [17]. 3. Results and discussion 3.1. Chloride inhibits the aminoacylation reaction Fig. 1 shows the e¡ect of KCl and Tris-HCl bu¡er on tyrosyl-tRNA synthesis when the magnesium concentration is varied. The physiological free [Mg2 ] of 1 mM is marked with a dashed line (as in other ¢gures). The chloride inhibition is observed below about 3 mM free [Mg2 ] but above this value no big changes in the activities are found. The e¡ect of 150 mM Tris bu¡er (125 mM Cl3 ) is still somewhat stronger than of 50 mM Tris bu¡er+100 mM KCl. Potassium acetate (100 mM) was present to keep the [K ] high. Sulphate also strongly inhibited. 50 mM K2 SO4 caused the same e¡ect as 150 mM KCl. Potassium acetate did not cause inhibition (below 200 mM), although it lies between sulphate and chloride in the Hofmeister anion series [18]. HEPES anion (25^ 125 mM) in the HEPES-KOH bu¡er was also without inhibiting e¡ect. The salt e¡ect is a sum of di¡erent e¡ects such as activation by potassium ions at concentrations below 100 mM and inhibition by chloride (or sulphate) at low Mg2 concentrations. If 100 mM K-acetate was added to keep the K concentration high, even low chloride concentrations ( 6 50 mM) were inhibitory, but without K-acetate, 50 mM KCl slightly activated when the K activation was stronger than the Cl3 inhibition. The apparent Michaelis constants for tRNATyr were strongly increased by KCl. The Hanes plots were essentially straight lines (at 1 mM free Mg2 , 2 mM MgATP, 1 WM Tyr). The Kapp m (tRNA) values were 22, 37, 93 and 240 nM at 0, 50, 100, and 150 mM KCl, respectively. 3.2. Chloride e¡ects on the PPi inhibition Chloride strengthened the PPi inhibition at all [Mg2 ] values (Fig. 2), although the apparent chlo-
ride inhibition was observed at low magnesium concentrations. The Kapp i (PPi ) values at 1 mM free magnesium were 140, 120, and 56 WM at 0, 50, and 150 mM KCl, respectively. 50 mM K2 SO4 caused almost exactly the same e¡ect as 150 mM KCl in Fig. 2C. In general, the PPi inhibition is strengthened if the dissociation constant for PPi is lowered, or if the pyrophosphorolysis rate k33 is increased, or if the rate forwards in the next steps are lowered. 3.3. Chloride e¡ects on the AMP inhibition AMP inhibits the aminoacylation typically with values of about 0.3^0.7 mM. Chloride had almost no e¡ect on the AMP inhibition at high (8 mM free) magnesium concentrations but at low (below 1 mM free) magnesium concentrations the inhibition was clearly weakened (Fig. 3). The Kapp i (AMP) values at 1 mM free magnesium were 0.35, 0.5, and 0.9 mM at 0, 50, and 150 mM KCl, respectively. The AMP inhibition is weakened if the dissociation constant (K7 ) of AMP is increased or if the reverse transfer reaction (k36 ) is lowered or if the rate of the dissociation of the aminoacyl-tRNA from the enzyme is increased. Weaker e¡ects on the inhibition may come from other parts of the total reaction. The parallel shift upwards of the plot at 1.5 mM Mg2 with increasing KCl concentrations can be simulated by increasing the value of KME4 . (The parallel shift is stronger in B.s.TyrRS than in E.c.TyrRS.) Kapp i
3.4. Some details of the mechanism Fig. 4 shows some kinetic experiments used in designing the detailed mechanism for the best-¢t analysis. The KTyr ( = K2 ) value from the intersection point in Fig. 4A is 19 WM. In Fig. 4B a sharp peak at about 0.5 mM Mg2 free is observed. The fall in the ATP/PPi exchange rate above this magnesium concentration can be almost totally explained by the decrease in the [MgPPi ] by formation of Mg2 PPi . The reactive species is MgPPi (e.g. due to the microscopic reversibility when MgATP is the reactive compound in the forward reaction) and the Kd value for the dissociation of one magnesium from Mg2 PPi is as low as 2.6 mM [19]. In the absence of tRNA an additional inhibiting binding of magnesium in seg-
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Fig. 4. Some kinetic experiments on TyrRS from E. coli. (A) Hanes plots of the aminoacylation reaction when [Tyr] and [MgATP] were varied. (B) Dependence of the ATP/PPi exchange rate on magnesium, AMP, and tRNA concentrations. (C) PPi dependence of the relation of the rates of the ATP/PPi exchange and aminoacylation of tRNA at identical conditions. [Mg2 ]total was 3 mM+[PPi ]. (D) Hanes plots of the ATP dependence of the aminoacylation reaction and inhibition by AMP. The concentration of Mg(acetate)2 was 10 mM+[ATP].
ment 2 improves the ¢t. In the presence of tRNA this inhibiting binding of magnesium is very weak and in the presence of chloride not observed. Therefore it was not taken into account in Eq. 3 nor in the analyses of Figs. 2 and 3. In Fig. 4B the exchange rate is reduced in the presence of tRNA because the enzyme is mainly in the late intermediates (segments 3 and 4 in Scheme 2) and thus not available in the exchange reaction. In Fig. 4C the relation vexchange /vacylation was measured. The kinetic equations for the relation are very
simple: if the conformational change (k6C ) is not included vexch /vacyl = C21 /C24 [12], and if it is included, vexch /vacyl = (C21 /C23 )U(C34 +C32 )/C34 . (The terms are from Scheme 2.) The expressions contain only a limited amount of rate and equilibrium constants and therefore it is useful in an estimation of the detailed mechanism. Straight lines in Fig. 4C were expected. The clearly curved plots were ¢tted only if both PPi and tRNA could be bound to the enzyme during the conformational-change step. The value of vexch /vacyl Tyr (at 1 mM Mg2 , 100 WM PPi ) was free , 1 WM tRNA
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about 1 in Fig. 4C but at optimal conditions the rate of the ATP/PPi exchange (without tRNA) was 8.7 times higher than the aminoacylation rate (without PPi ). The e¡ect of AMP on the Hanes plots for the ATP dependence of the aminoacylation (Fig. 4D) can be used when the events after the transfer reaction are analysed. The intersection on the ordinate axis would mean an uncompetitive inhibition mechanism (from segment 4). A competitive inhibition (from segment 1) would give parallel lines. Curved plots suggest a second, activating binding of ATP. Fig. 4D shows that the AMP inhibition almost totally comes from segment 4.
b
b
b
3.5. Best-¢t analysis The principle of `global analysis' [17] was applied, i.e. the same detailed reaction mechanism and the same constant values were used for several di¡erent experiments. The main purpose was to ¢nd the correct mechanism, which includes testing the possible steps, the relative rates of the steps and which ligands are bound at a given step and in which order. If the mechanism is correct, the error rates in the analyses of all di¡erent experiments should be low. If the error of the assay (sxWy of the rate curve) is 2^3%, errors of 3^5% are attained with a good mechanism but a weak mechanism gives errors of tens of per cent. Over 10% average errors were regarded as non-acceptable. Simultaneous analysis of several, chosen, experiments is an e¤cient way to reject wrong mechanisms and to ¢nd the steps of the complex reaction in which the observed features are connected. The same is not true if the best-¢t analysis is based on only one experiment where two ligand concentrations are varied. The following list gives the details of the mechanism and the reasons for their use: b
b
Only one Mg2 is assumed to be bound to tRNA to make it reactive in the transfer reaction. Several magnesium ions are known to be bound to tRNA [20], but in the simplest model the present system behaves as if only one Mg2 would be responsible for the structural change. The concentrations of di¡erent forms of ATP and PPi (free ATP, MgATP, free PPi , MgPPi , Mg2 PPi )
b
b
b
are calculated for every measurement point using the Kd values, and so they are taken into account in the analysis. The amino acid is bound to the enzyme before ATP [21]. Both the random model and the model where two ATP molecules are required were weaker in the best-¢t analysis, especially when measured by the ATP/PPi exchange. tRNA and ATP do not a¡ect the binding of each other. This is not necessarily true, probably tRNA and ATP weaken the binding of each other, but the assumption was made for simplicity since it has only a very weak e¡ect in the experiments of this work. AMP can be bound to the ATP site before the activation reaction. The e¡ect of this binding, however, is very weak (Fig. 4D), the stronger inhibition comes from the steps after the transfer reaction. A conformational change between the activation and transfer steps is included in the mechanism, as earlier [13]. It is a reorganization of the active site for the transfer reaction, and the change can happen in the enzyme, tRNA or both. Such changes are observed in the crystal structures when tRNA is bound [22^25] but the existence in the kinetic mechanism has not been self-evident. The present data, the chloride e¡ects, could not be satisfactorily ¢tted if this step was omitted. Some data (e.g. the curvature in Fig. 4C) required that the above conformational change can occur also when PPi is bound and also with both free tRNA and MgtRNA. In the transfer reaction PPi is not bound and MgtRNA is required. The dissociation of the aminoacyl-tRNA from the enzyme is enhanced by two events: dissociation of the magnesium ion from the enzymeWaa-tRNA complex [13] and binding of ATP to the complex (maybe to the AMP site) [12]. Both the experiments in Fig. 4D and the best ¢t analysis of the chloride e¡ect strongly support this model. Although the gross model shows the involvement of the two events, the details remain to be solved. A rate-limiting step at the end of the total aminoacylation reaction has often been detected with di¡erent aminoacyl-tRNA synthetases [26]. The slow step (k8 , k8A , k8AMP ) in the equations is thought to be a conformational change (maybe
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Table 1 Dependences of some kinetic constants on [KCl] [KCl] (mM)
KMR (mM)
KME4 (mM)
K5M (WM)
K5 (WM)
0 50 150
0.11 þ 0.05 0.3 þ 0.06 2.8 þ 0.8
0.6 þ 0.14 2.5 þ 1.2 V13
0.42 þ 0.12 0.5 þ 0.1 0.6 þ 0.14
1.2 1.4 4.5
KMR is Kd of Mg2 from MgtRNA, KME4 is Kd of Mg2 from the complex enzymeWTyr-tRNAWMg, K5M is Kd of MgtRNA from enzymeWMgtRNA, and K5 is Kd of tRNA from (non-reactive) enzymeWtRNA.
corresponding to that with k36C ) which precedes a rapid dissociation of the product.
The detailed kinetic mechanism of the chloride effect is not known and was not written into the equations. The di¡erent chloride concentrations were analysed separately. Three di¡erent experiments were included in the `global'-type analysis, and the sum of the error percentages was minimized when the constant values were optimized. The experiments were the PPi inhibition (Fig. 2), the AMP inhibition (Fig. 3), and the Kapp m (tRNA) assay. Very simple changes in the constant values are required to obtain an excellent ¢t (3^5% error) at di¡erent KCl concentrations (Table 1), and all these changes are involved in the binding or dissociation of tRNA from the enzyme. The minimal changes for a satisfactory ¢t (5^6% average error) would be the increases only in the KMR and KME4 values. (These two constants may represent the same event, dissociation of Mg2 from MgWtRNA, at di¡erent steps of the total reaction.) Table 1 also shows that in the best-¢t system there is almost no change with increasing [KCl] in the real dissociation constant, K5M , of the reactive species MgtRNA. The big change in the Kapp m (tRNA) value comes through the weakened binding of Mg2 to tRNA. 3.6. Comparison of the tyrosyl-tRNA synthetases from E. coli and B. stearothermophilus The kinetic mechanism of the TyrRS from B. stearothermophilus has been thoroughly studied previously [17,27^29]. When the constant values and mechanism of those works were applied in the best¢t analyses of the present work, not even a close ¢t was attained. The minimum errors were tens of per cents. Major changes both in the mechanism and in
values of some individual constants were required. Therefore some kinetic experiments were also done with the TyrRS from B. stearothermophilus (Fig. 5). The similarity is striking between the results in Fig. 5 and the corresponding or relating experiments with the E. coli enzyme in Figs. 1 and 4. The experiments show that there are no big di¡erences in the mechanisms of TyrRS:s from the two bacteria. The relation vexch /vacyl was lower in B.s.TyrRS than in E.c.TyrRS. However at optimal conditions the rate of the ATP/ PPi exchange (without tRNA) was 7.3 times higher than the aminoacylation rate (without PPi ), thus the relation is not essentially di¡erent from the value of 8.7 for the E.c.TyrRS. The details of the strong reduction of the ATP/PPi exchange rate of B.s.TyrRS by tRNA have not yet been analysed. The analysis of the KCl e¡ects on the PPi inhibition and AMP inhibition showed that the KCl e¡ect was somewhat stronger with the B. s. enzyme (with tRNA from E. 2 coli). The Kapp i (PPi ) values (at 1 mM [Mg ]free ) were app 230, 100, and 48 WM, and the Ki (AMP) values were 0.32, 0.50, and 2.15 mM at 0, 50, and 150 mM KCl, respectively. Some di¡erences existed in the measurement conditions between the previous studies with B. s. TyrRS and the present work: typically in the previous studies the chloride concentration was about 130 mM (100 mM from the bu¡er, 30 mM from MgCl2 ), [Mg2 ]free was 10 mM, and the pH of the Tris-HCl bu¡er was 7.78 [17,28], while in the present work no Cl3 , about 1 mM free Mg2 , and the HEPES-KOH bu¡er pH 7.4 were used. The di¡erences in the chloride and magnesium concentrations seem to be su¤cient to explain most of the observed di¡erences. If high chloride concentrations are used, high Mg2 concentrations must be used for optimal activity (Fig. 1). If high magnesium concentrations are also systematically used in the presence of PPi in the measurements of pyrophosphorolysis or ATP/PPi ex-
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Fig. 5. Some kinetic experiments on TyrRS from B. stearothermophilus. A is like Fig. 1A, D like Fig. 1B, B like Fig. 4A, C like Fig. 4B. E is like Fig. 4C but [Mg2 ] is varied instead of [tRNA]. F is like Fig. 4C but [Mg2 ]total is 1 mM+[ATP].
change, most of PPi is in the form of Mg2 PPi , while MgPPi would be the reactive compound. From the Kd value (2.6 mM), 78% of PPi is in the form of Mg2 PPi at 10 mM free Mg2 . The lowered concentration of the reactive species MgPPi should be taken into account in the calculations of the pyrophosphorolysis rates and kinetic constants through the ATP/ PPi exchange measurements, but in the works mentioned above it has not been taken into account. In the earlier studies [17] with B.s.TyrRS some constant values were k33 = 16.6 s31 , KPP = 0.61 mM, and KATP = 4.7 mM, but the corresponding values in the present work were 120 s31 , 0.23 mM and 0.45 mM. The di¡erences in k33 and KPP can be explained by the presence of Mg2 PPi , but the reason for the big di¡erence in the KATP value remains obscure. The low Kapp m (ATP) value from Fig. 5F (160 WM) would support the low KATP value. The analyses of the previous studies did not include a slow step after
the transfer reaction, but the present data could not be ¢tted without the slow step. 3.7. Nature of the chloride e¡ect The molecular basis of the lowered a¤nity between the enzyme and tRNA in the presence of chloride (or sulphate) is not known. The chloride e¡ect resembles the other cases of protein-nucleic acid interactions where replacement of chloride by acetate or glutamate strengthens the interaction [2^ 4]. For instance, the complex formation between RNA polymerase and the VPR promoter is a multistep process where chloride favours one conformation but Mg2 another, transcriptionally competent, conformation [4]. In an analogous way, chloride may favour a non-reactive conformation of TyrRS or the CCA end of tRNA, but binding of Mg2 causes the formation of the reactive conformation.
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It looks like the e¡ect in the present work would not be principally caused by salt concentration since K-acetate does not inhibit at the concentrations used. Hofmeister anion e¡ect on the water surface of the macromolecules is also not an obvious explanation. More speci¢c e¡ects of the anions may be necessary. Sulphate, acetate and chloride belong to the stabilizing end of the Hofmeister series [18] but of these sulphate is the strongest inhibitor and has the strongest e¡ect on the PPi inhibition while acetate has almost no e¡ect. 3.8. On the enzyme assays Most assays of the aminoacyl-tRNA synthetases have been done in Tris-HCl bu¡ers and potassium chloride has been added to increase the K concentration. The present results suggest that these conditions should be reconsidered. In normal assays of the aminoacylation activity the e¡ect of high chloride concentration is reversed at high Mg2 concentrations, and therefore is not serious (at least in TyrRS). However, when reaction mechanisms including binding of tRNA are studied, or if PPi is used in the measurements, the e¡ects of high chloride and magnesium concentrations should be taken into account. Furthermore, MgCl2 or MgSO4 should not be used in the studies of the Mg2 e¡ects since the interacting anion concentration is changed at the same time. Chloride can also have some e¡ects in the measurements of the proofreading since the correction systems are placed between the binding of the tRNA and dissociation of the aa-tRNA. References [1] M.T. Record Jr., W. Zhang, C.F. Anderson, Adv. Protein Chem. 51 (1998) 281^353. [2] S. Leirmo, C. Harrison, D.S. Cayley, R.R. Burgess, M.T. Record Jr., Biochemistry 26 (1987) 2095^2101. [3] J.-H. Ha, M.W. Capp, M.D. Hohenwalter, M. Baskerville, M.T. Record Jr., J. Mol. Biol. 228 (1992) 252^264.
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[4] W.C. Suh, S. Leirmo, M.T. Record Jr., Biochemistry 31 (1992) 7815^7825. [5] R.B. Loft¢eld, E.A. Eigner, J. Biol. Chem. 242 (1967) 5355^ 5359. [6] R.B. Loft¢eld, E.A. Eigner, A. Pastuszyn, T.N.E. Lo«vgren, H. Jakubowski, Proc. Natl. Acad. Sci. USA 77 (1980) 3374^ 3378. [7] D.W.E. Smith, J. Biol. Chem. 244 (1969) 896^901. [8] M. Yarus, Biochemistry 11 (1972) 2050^2060. [9] E. Kellenberger, in: F.C. Neidhardt (Ed.), Escherichia coli and Salmonella. Cellular and Molecular Biology, ASM Press, Washington, DC, 1996, pp. 17^28. [10] T. Alatossava, H. Ju«tte, A. Kuhn, E. Kellenberger, J. Bacteriol. 162 (1985) 413^419. [11] R.K. Airas, Eur. J. Biochem. 192 (1990) 401^409. [12] R.K. Airas, Eur. J. Biochem. 210 (1992) 443^450. [13] R.K. Airas, Eur. J. Biochem. 240 (1996) 223^231. [14] S. Cha, J. Biol. Chem. 243 (1968) 820^825. [15] M.L. Johnson, L.M. Faunt, Methods Enzymol. 210 (1992) 1^37. [16] J.M. Beechem, Methods Enzymol. 210 (1992) 37^54. [17] D.M. Lowe, G. Winter, A.R. Fersht, Biochemistry 26 (1987) 6038^6043. [18] M.G. Cacace, E.M. Landau, J.J. Ramsden, Q. Rev. Biophys. 30 (1997) 241^277. [19] S.E. Volk, A. Baykov, V.S. Duzhenko, S.M. Avaeva, Eur. J. Biochem. 125 (1982) 215^220. [20] D.C. Lynch, P.R. Schimmel, Biochemistry 13 (1974) 1841^ 1852. [21] P. Brick, T.N. Bhat, D.M. Blow, J. Mol. Biol. 208 (1988) 83^98. [22] S. Cusack, A. Yaremchuk, M. Tukalo, EMBO J. 15 (1996) 2834^2842. [23] S. Cusack, A. Yaremchuk, M. Tukalo, EMBO J. 15 (1996) 6321^6334. [24] B. Rees, J. Cavarelli, D. Moras, Biochimie 78 (1996) 624^ 631. [25] M.A. Rould, J.J. Perona, D. So«ll, T.A. Steitz, Science 246 (1989) 1135^1142. [26] P.R. Schimmel, D. So«ll, Annu. Rev. Biochem. 48 (1979) 601^648. [27] A.R. Fersht, J.W. Knill-Jones, H. Bedouelle, G. Winter, Biochemistry 27 (1988) 1581^1587. [28] T.N.C. Wells, J.W. Knill-Jones, T.E. Gray, A.R. Fersht, Biochemistry 30 (1991) 5151^5156. [29] J.M. Avis, A.G. Day, G.A. Garcia, A.R. Fersht, Biochemistry 32 (1993) 5312^5320. [30] E.A. Galinski, Adv. Microb. Physiol. 37 (1996) 273^328.
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