Comparison of the Catalytic Roles Played by the KMSKS Motif in the Human and Bacillus stearothermophilus Tyrosyl-tRNA Synthetases*

The Class I aminoacyl-tRNA synthetases are characterized by two signature sequence motifs, “HIGH” and “KMSKS.” In Bacillus stearothermophilus tyrosyl-tRNA synthetase, the KMSKS motif (230KFGKT234) has been shown to stabilize the transition state for tyrosine activation through interactions with the pyrophosphate moiety of ATP. In most eukaryotic tyrosyl-tRNA synthetases, the second lysine in the KMSKS motif is replaced by a serine or an alanine residue. Recent kinetic studies indicate that potassium functionally compensates for the absence of the second lysine in the human tyrosyl-tRNA synthetase (222KKSSS226). In this paper, site-directed mutagenesis and pre-steady state kinetics are used to determine the roles that serines 224, 225, and 226 play in catalysis of the tyrosine activation reaction. In addition, the catalytic role played by a downstream lysine conserved in eukaryotic tyrosyl-tRNA synthetases, Lys-231, is investigated. Replacing Ser-224 and Ser-226 with alanine decreases the forward rate constant 7.5- and 60-fold, respectively. In contrast, replacing either Ser-225 or Lys-231 with alanine has no effect on the catalytic activity of the enzyme. These results are consistent with the hypothesis that the KMSSS sequence in human tyrosyl-tRNA synthetase stabilizes the transition state for the tyrosine activation reaction by interacting with the pyrophosphate moiety of ATP. In addition, although they play similar roles in catalysis, the overall contribution of the KMSKS motif to catalysis appears to be significantly less in human tyrosyl-tRNA synthetase than it is in the B. stearothermophilus enzyme.

AARS ϩ AA ϩ MgATP^AARS⅐AA Ϫ AMP ϩ PP i (Eq. 1) AARS⅐AA Ϫ AMP ϩ tRNA AA^A ARS ϩ AA Ϫ tRNA AA ϩ AMP (Eq. 2) The Class I aminoacyl-tRNA synthetase family, of which tyrosyl-tRNA synthetase is a member, is characterized by the presence of an amino-terminal Rossmann-fold catalytic domain and conserved HIGH and KMSKS signature sequences (1)(2)(3)(4)(5)(6)(7)(8)(9). The KMSKS signature sequence in the Bacillus stearothermophilus tyrosyl-tRNA synthetase ( 230 KFGKT 234 ) participates in catalysis of the tyrosine activation reaction (10 -15). Specifically, Lys-230, Lys-233, and Thr-234 stabilize the transition state by interacting with the pyrophosphate moiety of the ATP substrate (11)(12)(13)(14)(15). In the human tyrosyl-tRNA synthetase Gly-232, Lys-233, and Thr-234 are replaced with serine residues ( 222 KMSSS 226 ) (16). The absence of a second lysine in the KMSSS sequence in human tyrosyl-tRNA synthetase, which is the most highly conserved amino acid in the Class I aminoacyl-tRNA synthetase family (17), and the observation that the catalytic efficiency of human tyrosyl-tRNA synthetase is similar to that of the B. stearothermophilus enzyme (18) raises the question of how human tyrosyl-tRNA synthetase compensates for the absence of the second lysine in the KMSKS signature sequence. Recently, it was shown that in the human tyrosyl-tRNA synthetase, potassium stabilizes the transition state for tyrosine activation by interacting with the pyrophosphate moiety of ATP (19). Based on these observations, it was concluded that in human tyrosyl-tRNA synthetase, potassium functionally replaces the second lysine in the KMSKS signature sequence. In this paper, site-directed mutagenesis and pre-steady state kinetic methods are used to determine whether other amino acids in the KMSSS loop of human tyrosyl-tRNA synthetase help compensate for the absence of the second lysine. Specifically, the roles that the three serine residues in the KMSSS sequence, Ser-224, Ser-225, and Ser-226, play in the catalysis of tyrosine activation are investigated. In addition, the hypothesis that a downstream lysine residue, Lys-231, which is conserved in eukaryotic tyrosyl-tRNA synthetases, also helps compensate for the absence of the second lysine in the KMSKS signature sequence is tested. The results presented in this paper indicate that of these four residues only Ser-224 and Ser-226 are involved in catalysis of the tyrosine activation reaction. Furthermore, the dissociation constant for potassium is not affected by replacing the serine residues in the KMSSS sequence of human tyrosyl-tRNA synthetase with alanine. These observations are consistent with the hypotheses that the KMSSS sequence in human tyrosyl-tRNA synthetase stabilizes the transition state for tyrosine activation through interactions with the pyrophosphate moiety of ATP. Quantitative analysis of tyrosyl-tRNA synthetase variants indicates that, although the KMSKS signature sequences play similar roles in the catalytic mechanisms of the B. stearothermophilus and human tyrosyl-tRNA synthetases, the extent to which the KMSSS sequence affects catalysis in the human enzyme is significantly less than that of the homologous sequence in the B. stearothermophilus tyrosyl-tRNA synthetase. This difference in the catalytic mechanisms of the B. stearothermophilus and human tyrosyl-tRNA synthetases potentially could be used to design inhibitors that selectively target the bacterial enzyme.
Site-directed Mutagenesis, Purification of the Wild-type and Variant Human Tyrosyl-tRNA Synthetases, and Standard Buffer-All tyrosyl-tRNA synthetase variants were created by the PCR-mediated overlapextension mutagenesis method (20). The pHYTS3-wt plasmid, a derivative of the pET30a(ϩ) cloning vector containing the wild-type human tyrosyl-tRNA synthetase gene (16), was used as the template for the initial PCR mutagenesis reactions. The following oligonucleotides were used to introduce the desired mutations and to remove a One additional primer, 5Ј-CA GAA GAG GAG TCC AAG ATT GAT CTC CTT GAT-3Ј, was used as an internal primer, and T7 promoter and T7 terminator primers were used as outside primers. The removal of the SacI site did not alter the amino acid sequences of the final products. All of the PCR products were purified with the Wizard PCR prep DNA Purification and DNA Cleanup Systems from Promega. The final PCR products were digested with the KpnI and HindIII restriction enzymes and subcloned into the pET30a(ϩ) vector that had been digested with the same two restriction enzymes. Positive clones were selected by PCR amplification of the human tyrosyl-tRNA synthetase coding sequence followed by digestion with the SacI restriction enzyme to ensure that the SacI site was removed. The tyrosyl-tRNA synthetase coding sequence of each variant was then sequenced by automated DNA sequencing to ensure that no secondary mutations had occurred. Purification of the wild-type and variant human tyrosyl-tRNA synthetases has been described previously (16,18). Briefly, the wild-type and tyrosyl-tRNA synthetase variants were expressed with a removable amino-terminal His tag/S tag. The cells were lysed by sonication in buffer A (50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, and 1 mM phenylmethanesulfonyl fluoride, pH 8), and the cellular debris was removed by centrifugation. The cleared lysate was then loaded onto a nickel-nitrilotriacetic acid gravity flow column equilibrated with buffer A, and the human tyrosyl-tRNA synthetase was eluted with buffer A containing 250 mM imidazole. The peak fractions were pooled and dialyzed against buffer B (20 mM Tris, pH 7.5, 0.1 mM EDTA, and 10 mM ␤-mercaptoethanol) plus 0.1 mM tetrasodium pyrophosphate overnight to remove any bound tyrosyladenylate, followed by dialysis against buffer B. The protein solution was then loaded onto a BioRad UNO Q6 anion exchange high pressure liquid chromatography column and eluted with a gradient of 0 -1 M NaCl in buffer B. The peak eluting at 140 mM NaCl was collected and dialyzed against two changes of buffer C (50 mM Tris, pH 7.5, 20 mM ␤-mercaptoethanol, and 10 mM MgCl 2 ) and finally against buffer C plus 10% glycerol (v/v). SDS-PAGE analysis was used to assess the purity of the purified enzymes (21). The concentration of the enzyme was determined using a filter-based active site titration assay as described in Ref. 22 and from A 280 measurements in the presence of 6 M guanidine hydrochloride (⑀ ϭ 89040 M Ϫ1 cm Ϫ1 as determined by the ExPASy ProtParam tool (23) (24,25).
Kinetic Analyses-All kinetic analyses were performed in the standard buffer Ϯ KCl at 25°C. Stopped-flow fluorescence studies were used to monitor the ATP dependence of tyrosyl-adenylate formation (i.e. the forward reaction) and the pyrophosphate dependence of the reverse reaction (i.e. the conversion of E•Tyr-AMP ϩ PP i to E ϩ Tyr ϩ ATP) in the pre-steady-state (26). In these studies an SX 18.MV stopped-flow spectrophotometer (Applied Photophysics) was used to monitor the decrease in fluorescence of the human tyrosyl-tRNA synthetase associated with the formation of the TyrRS 1 •Tyr-AMP complex and the increase in fluorescence associated with the reverse reaction ( ex ϭ 295 nm, em Ͼ 320 nm) (18) (18,26). Due to the low solubility of MgPP i , it is not possible to achieve saturating pyrophosphate concentrations for the reverse reaction. As a result, it was not possible to determine k -3 independently of K PP i . Instead, the reverse reaction was monitored under conditions where K PP i Ͼ Ͼ [PP i ] and k -3 /K PP i were determined by fitting the data to the resulting linear approximation of Equation 5. In the above calculations, a rapid equilibrium assumption is used for the binding of the substrates (18). All kinetic data were fit to a single exponential floating end point equation using the Kaleidagraph and Applied Photophysics stoppedflow software packages to determine the observed rate constants. The Kaleidagraph software was used to plot the observed rate constants versus the substrate concentrations and to fit these plots to the following hyperbolic function (Equation 5) where k obs is the observed rate constant, k i is the either the forward rate constant (k 3 ) or the reverse rate constant (k -3 ) for the tyrosine activation reaction, [S] T is the total substrate concentration, and K d is the disso-ciation constant of the substrate of interest (24). The data were also subjected to the Eadie-Hoftsee linear transformation to determine how well the data fit the above model. Calculation of Standard Free Energy Levels-The standard free energies for each state along the reaction pathway were calculated from the rate and dissociation constants using the following equations (Equations 6 -10) and assuming standard states of 1 M for ATP, tyrosine, and pyrophosphate: where G o is the Gibbs standard free energy, R is the gas constant, T is the absolute temperature, k B is the Boltzmann constant, h is Planck's constant, and [TyrRS•Tyr-ATP] ‡ is the transition state complex (27). The standard free energies for each complex in Equations 6 -10 are calculated relative to the standard free energy of the unliganded enzyme (⌬G TyrRS o ). The Gibbs activation energy for the formation of tyrosyl-adenylate was calculated by taking the difference in standard free energies between the transition state and the TyrRS•Tyr•ATP complex that immediately precedes the transition state.

Determination of the Dissociation Constant for Tyrosine
(K Tyr )-The reaction mechanism for tyrosine activation is shown in Scheme I. To determine whether serines 224, 225, 226, and lysine 231 interact with the tyrosine substrate, alanine variants at each of these positions were generated and the dissociation constant for tyrosine in the absence of ATP (K Tyr ) was determined for each variant using equilibrium dialysis. As shown in Table I, each of the variants bound tyrosine with an affinity similar to that of the wild-type enzyme (34 M).
Determination of the Dissociation Constant of ATP (KЈ ATP ) and the Forward Rate Constant (k 3 )-To determine whether serines 224, 225, 226, and lysine 231 interact with the ATP substrate and/or are involved in catalysis of the tyrosine activation reaction, alanine variants at each of these positions were analyzed using pre-steady state kinetic methods. The dissociation constant for ATP in the presence of saturating tyrosine (KЈ ATP ) and the forward rate constant (k 3 ) were determined from the ATP dependence of the tyrosine activation reaction in the presence of saturating tyrosine (200 M). As shown in Fig. 1 and summarized in Table I, KЈ ATP for these variants is similar to that observed for the wild-type enzyme. In contrast, the forward rate constant for tyrosine activation (k 3 ) is reduced 8-and 60-fold for the S224A and S226A variants, respectively. The S225A and K231A substitutions display forward rate constant values similar to that of the wild-type enzyme.
Determination of the Specificity Constant for the Reverse Reaction (k -3 /K PP i )-Pre-steady state kinetic analyses of the reverse reaction for the S224A, S225A, and S226A variants were performed. Due to the low solubility of MgPP i , it is not possible to determine the reverse rate constant (k -3 ) and the dissociation constant for pyrophosphate (K PP i ) independently of each other. Under conditions where [PP i ] Ͻ Ͻ K PP i , however, Equation 5 can be approximated by a linear equation whose slope is given by the specificity constant (k -3 /K PP i ). As a result, even though k -3 and K PP i cannot be determined independently of each other, the specificity constant can be determined by fitting a plot of k obs versus [PP i ] to a linear equation. As shown in Fig. 2 and summarized in Table I, k -3 /K PP i is reduced ϳ12and 100-fold for the S224A and S226A variants, respectively, whereas the S225A substitution has only a small effect on the value of k -3 /K PP i . Due to its lack of effect on K Tyr , KЈ ATP , and k 3 , the specificity constant for the reverse reaction was not determined for the K231A variant.
Potassium Does Not Interact with the Serine Residues of the KMSSS Sequence-Unlike bacterial tyrosyl-tRNA synthetases, tyrosine activation by the human tyrosyl-tRNA synthetase is potassium-dependent. To determine whether potassium interacts with the serine residues in the KMSSS sequence, pre-steady state kinetic methods were used to determine whether the alanine variants display altered affinities for potassium relative to that of the wild-type enzyme. The dissociation constant for potassium was determined from the potassium dependence of the tyrosine activation reaction in the presence of saturating tyrosine (200 M) and ATP (10 mM). All three of the variants bind potassium with affinities similar to that observed for the wild-type tyrosyl-tRNA synthetase, indicating that the KMSSS sequence is not involved in binding the potassium ion (Fig. 3, Table I).

Free Energy Calculations for Each
Step Along the Reaction Pathway-The binding and rate constants summarized in Table I were used to calculate the standard free energy values for each step along the reaction pathway for the S224A, S225A, S226A, and K231A variants. For the S225A and K231A variants, the standard free energies of each complex are similar to those observed for the wild-type enzyme. For this reason, only the free energy profile for the S225A variant is shown (Fig. 4,  B). In contrast, the standard free energy values of the transition state complexes for the S224A and S226A variants are 1.5 and 2.6 kcal/mol higher, respectively, than the value for the wild-type enzyme (Fig. 4, A and C). In addition, the activation energy is increased 1.3 and 2.4 kcal/mol by the S224A and S226A substitutions, respectively. The results of the S226A substitution are similar to those observed for the T234A substitution in the B. stearothermophilus enzyme, which increases the free energy for the transition state by 2.8 kcal/mol and increases the activation energy by 3.8 kcal/mol (14). DISCUSSION The KMSSS Sequence Catalyzes the Formation of Tyrosyl-Adenylate by Interacting with the Pyrophosphate Moiety of ATP-In the B. stearothermophilus tyrosyl-tRNA synthetase 230 KFGKT 234 sequence, three residues, Lys-230, Lys-233, and Thr-234, stabilize the transition state for tyrosine activation by interacting with the pyrophosphate moiety of the ATP substrate (11)(12)(13)(14)(15). In the human tyrosyl-tRNA synthetase, Lys-230 is conserved, whereas Gly-232, Lys-233, and Thr-234 are replaced with Ser-224, Ser-225, and Ser-226, respectively (16). Previous studies indicate that Gly-232 in the B. stearothermophilus tyrosyl-tRNA synthetase can be replaced by alanine without significantly destabilizing the transition state complex (12). The results presented in this study indicate that replacement of the corresponding residue in the human tyrosyl-tRNA synthetase, Ser-224, by alanine destabilizes the transition state for the tyrosine activation reaction by 1.5 kcal/mol. The observation that the S224A substitution does not affect the stabilities of the TyrRS•Tyr, TyrRS•Tyr•ATP, or TyrRS•Tyr-AMP complexes suggests that Ser-224 interacts with the pyrophosphate moiety of the ATP substrate and that this interac-SCHEME 1 tion only occurs during formation of the transition state. 2 The second lysine in the KFGKT sequence, Lys-233, in the B. stearothermophilus tyrosyl-tRNA synthetase is replaced by a serine, Ser-225, in the human enzyme. In B. stearothermophilus tyrosyl-tRNA synthetase, replacing lysine 233 with alanine, introduces positive cooperativity into the enzyme with respect to the binding of ATP (13). It is postulated that this cooperativity is always present in B. stearothermophilus tyrosyl-tRNA synthetase but is only uncovered in the K233A variant due to the lower affinity of this variant for ATP. The addition of 0.5 M NaCl to the reaction mixture restores the high affinity binding of ATP to the active site and abolishes the cooperative kinetics (13). In the presence of NaCl, the primary effect of replacing Lys-233 with alanine is a 50-fold decrease in the forward rate constant. These results are consistent with the hypothesis that Lys-233 stabilizes the transition state for tyrosine activation primarily through interactions with the pyrophosphate moiety of ATP (13). Replacement of the equivalent residue in human tyrosyl-tRNA synthetase, Ser-225, with alanine has little effect on the catalytic activity.
In contrast to serines 224 and 225 in human tyrosyl-tRNA synthetase, whose roles differ from the corresponding residues in B. stearothermophilus tyrosyl-tRNA synthetase, the role of Ser-226 in the human enzyme is similar to its counterpart (Thr-234) in B. stearothermophilus tyrosyl-tRNA synthetase. Specifically, replacement of Ser-226 with alanine in the human enzyme results in a 60-fold decrease in the forward rate constant for tyrosine activation, whereas replacement of Thr-234 by alanine in B. stearothermophilus tyrosyl-tRNA synthetase decreases the forward rate constant for tyrosine activation by 540-fold (13). In addition, replacement of Thr-234 by alanine in B. stearothermophilus tyrosyl-tRNA synthetase increases the affinity of the enzyme for ATP 3-fold, whereas replacement of Ser-226 with alanine has little effect on the initial binding of ATP to the human tyrosyl-tRNA synthetase. Thus, although their roles are similar, Thr-234 is significantly more important in the catalytic mechanism of B. stearothermophilus tyrosyl-tRNA synthetase than its counterpart, Ser-226, is in the catalytic mechanism of the human enzyme. It is apparent from the kinetic analyses presented in this paper that, although the  KMSKS motifs in the human and B. stearothermophilus tyrosyl-tRNA synthetases play similar roles, the extent to which KMSSS catalyzes the formation of tyrosyl-adenylate in the human enzyme is significantly less than that of its counterpart in B. stearothermophilus tyrosyl-tRNA synthetase.
Potassium Does Not Interact with the KMSSS Sequence in the Human Tyrosyl-tRNA Synthetase-If the KMSSS sequence in human tyrosyl-tRNA synthetase is less important in catalysis than its counterpart in the B. stearothermophilus enzyme, why do the two enzymes display similar kinetic properties? Previous investigations indicate that the loss of catalytic function by the KMSSS sequence is compensated for by the involvement of potassium in the catalytic mechanism of human tyrosyl-tRNA synthetase (19). Specifically, potassium has been shown to functionally compensate for the absence of the second lysine in the KMSSS sequence (19). Like Ser-224 and Ser-226, potassium stabilizes the transition state for tyrosine activation by interacting with the pyrophosphate moiety of the ATP substrate. These observations raise the question of whether potassium interacts with the serine residues in the KMSSS signature sequence in the human tyrosyl-tRNA synthetase. The replacement of either of these serine residues with alanine did not decrease the binding affinity of the human tyrosyl-tRNA synthetase for potassium, indicating that the KMSSS sequence does not form part of the potassium-binding site.
Why Is the Second Lysine Absent from the KMSSS Sequence?-In eukaryotic tyrosyl-and tryptophanyl-tRNA synthetases, the second lysine in the KMSKS motif is replaced by either a serine or an alanine residue (28,29). Given the role that Lys-233 plays in the catalysis of tyrosine activation in the B. stearothermophilus enzyme it is interesting that it is not conserved in the human tyrosyl-tRNA synthetase. In addition, Lys-233, which corresponds to the second lysine of the KMSKS signature sequence, is the most highly conserved residue among all Class I aminoacyl-tRNA synthetases (17). The observation that its replacement in the human enzyme, Ser-225, does not play a significant role in the catalysis of tyrosine activation raises the question of why this variation occurs in nature. In particular, given the ability of this lysine to stabilize the transition state for tyrosine activation, it is curious that, with the exception of tyrosyl-tRNA synthetases in higher plants, no serine (or alanine) to lysine revertants have been observed in eukaryotic tyrosyl-and tryptophanyl-tRNA synthetases (Table II). This suggests that there may be groups in the eukaryotic tyrosyl-and tryptophanyl-tRNA synthetases that would interfere with the second lysine residue in the KMSKS motif. This hypothesis is currently being investigated.
Concluding Remarks-In this paper we provide evidence that the KMSSS sequence in human tyrosyl-tRNA synthetase decreases the forward rate constant for tyrosyl-adenylate formation by interacting with the pyrophosphate moiety of ATP. In addition, quantitatively comparing the kinetics for alanine variants at equivalent positions in the human and B. stearothermophilus tyrosyl-tRNA synthetases indicates that although the KMSKS motifs play similar roles in the two enzymes, the extent to which the KMSKS motif catalyzes the formation of tyrosyl-adenylate is significantly larger in the B. stearothermophilus enzyme than it is in the human enzyme. This is consistent with the observation that potassium appears to compensate for the absence of the second lysine in the KMSKS motif of the human tyrosyl-tRNA synthetase (19). The observation that the KMSKS motif plays a larger role in the catalytic mechanism of the B. stearothermophilus tyrosyl-tRNA synthetase suggests that inhibitors that interact with this mo-  Ser Ala a N/A indicates that the sequence was not available prior to publication of this paper. tif will have higher affinities for bacterial tyrosyl-tRNA synthetases than for their eukaryotic homologs.