Catalysis of Tyrosyl-Adenylate Formation by the Human Tyrosyl-tRNA Synthetase*

Although the active site residues in theBacillus stearothermophilus and human tyrosyl-tRNA synthetases are largely conserved, several differences exist between the two enzymes. In particular, three amino acids that stabilize the transition state for the activation of tyrosine in B. stearothermophilus tyrosyl-tRNA synthetase (Cys-35, His-48, and Lys-233) are not present in the human enzyme. This raises the question of whether the activation energy for the tyrosine activation step is higher for the human tyrosyl-tRNA synthetase than for the B. stearothermophilus enzyme. In this paper, we demonstrate that intrinsic fluorescence changes can be used to monitor the pre-steady state kinetics of human tyrosyl-tRNA synthetase. In contrast to theB. stearothermophilus enzyme, catalysis of the tyrosine activation step is potassium-dependent in the human tyrosyl-tRNA synthetase. Specifically, potassium increases the forward rate constant for tyrosine activation 260-fold in the human tyrosyl-tRNA synthetase. Comparison of the forward rate constants for catalysis of tyrosine activation by the human and B. stearothermophilus enzymes indicates that despite differences in their active sites and the potassium requirement of the human enzyme, the activation energies for tyrosine activation are identical for the two enzymes. The results of these investigations suggest that differences exist between the active sites of the bacterial and human tyrosyl-tRNA synthetases that could be exploited to design antimicrobials that target the bacterial enzyme.

Bacillus stearothermophilus tyrosyl-tRNA synthetase is a homodimeric enzyme that displays "half-of-the-sites" reactivity with respect to tyrosine binding and tyrosyl-adenylate formation (9 -11). Site-directed mutagenesis and pre-steady state kinetic analyses have been used to identify 18 active site amino acids that stabilize the transition state for tyrosine activation in the B. stearothermophilus enzyme (reviewed in Refs. 12 and 13). Four of these amino acids are absent in the human tyrosyl-tRNA synthetase. In the B. stearothermophilus enzyme, replacement of three of these amino acids, Cys-35, His-48, and Lys-233, destabilizes the transition state for tyrosine activation by 1.2, 1.2, and 3.0 kcal/mol, respectively (1)(2)(3)(4)(5)(6)14). His-48 and Lys-233 are members of the HIGH and KMSKS signature motifs that characterize the Class I aminoacyl-tRNA synthetases. These observations suggest that either the specific activity of the human tyrosyl-tRNA synthetase is less than that of the B. stearothermophilus enzyme, or alternate functional groups are present in the active site of the human tyrosyl-tRNA synthetase that compensate for the absence of Cys-35, His-48, and Lys-233. Identifying differences in the active sites of the human and bacterial tyrosyl-tRNA synthetases will facilitate the development of antibiotics that specifically target the bacterial enzyme.
In this paper, we show that a change in the intrinsic fluorescence of human tyrosyl-tRNA synthetase can be used to monitor the pre-steady state kinetics of tyrosine activation. Pre-steady state kinetic methods are then used to test the hypothesis that the human tyrosyl-tRNA synthetase is less active than the B. stearothermophilus enzyme with respect to tyrosine activation and to demonstrate that the human tyrosyl-tRNA synthetase requires potassium to stabilize the transition state complex during formation of the tyrosyl-adenylate intermediate (Scheme I).
Purification of the Recombinant Human Tyrosyl-tRNA Synthetase-Purification of the recombinant human tyrosyl-tRNA synthetase was carried out using a modification of the method described by Kleeman et al. (16). Briefly, the tyrosyl-tRNA synthetase was expressed with a removable amino-terminal His-tag/S-tag in Escherichia coli BL21DE3 pLysS cells harboring the pHYTS3-wt plasmid (16). 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 removed by centrifugation. The cleared lysate was then loaded onto a Ni-NTA 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 tyrosyl-adenylate, followed by dialysis against Buffer B. The protein solution was then loaded onto a Bio-Rad UNO Q6 anion exchange high performance 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 buffer 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 and Western blot analyses (using an antibody directed against the amino-terminal S-tag) were used to assess the purity of the human tyrosyl-tRNA synthetase (17,18). The concentration of the enzyme was determined using a filter-based active-site titration assay as described in Ref. 19 and from A 280 measurements in the presence of 6 M guanidine hydrochloride (⑀ ϭ 89,040 M Ϫ1 cm Ϫ1 as determined by the ExPASy ProtParam tool (20)). The enzyme was stored at Ϫ70°C.
Removal of the Amino-terminal His-tag-The amino-terminal Histag was removed by enterokinase digestion following the protocol in the Enterokinase Cleavage Capture Kit. Enterokinase digestion yields the wild type protein with no additional amino acids at the amino terminus. Enterokinase digestion was carried out overnight at 4°C with an enterokinase concentration of 5 units/mg of synthetase. Under these conditions the cleavage efficiency was 100%, with minimal (Ͻ20%) cleavage of the tyrosyl-tRNA synthetase at secondary sites. Following enterokinase digestion, the His-tag was then separated from the free enzyme with a second Ni-NTA affinity chromatography step followed by ion-exchange high performance liquid chromatography using the conditions described above.
Steady-state Emission Spectra of the Recombinant Human Tyrosyl-tRNA Synthetase-Steady-state fluorescence emission measurements were performed at 25°C using a TimeMaster Fluorescence Spectrometer (Photon Technology International). The intrinsic fluorescence of the human tyrosyl-tRNA synthetase, in Buffer D (150 mM Tris, pH 7.5, 150 mM KCl, 20 mM ␤-mercaptoethanol, 10 mM MgCl 2 ) plus 1 unit/ml pyrophosphatase, was measured in the absence and presence of substrates ( ex ϭ 295 nm, em ϭ 300 -400 nm). For the titration experiments, aliquots of either MgATP or tyrosine were added to the human tyrosyl-tRNA synthetase (4 M) or the B. stearothermophilus enzyme (2 M) and 1 unit/ml inorganic pyrophosphatase in either Buffer D alone, Buffer D ϩ 200 M tyrosine, or Buffer D ϩ 10 mM MgATP, respectively. Inorganic pyrophosphatase was added to the reaction to: 1) help drive the reaction in the direction of tyrosyl-adenylate formation; 2) prevent accumulation of the TyrRS 1 ⅐Tyr-AMP⅐PP i complex; and 3) prevent the reverse reaction from occurring. After allowing the reaction to equilibrate for 2 min at 25°C, the intrinsic fluorescence of the enzyme was then excited at 295 nm and the relative intensity of the fluorescence emission determined by integrating the area under the emission curve from 320 to 400 nm. The relative fluorescence intensity was normalized using the emission of the free enzyme as 100%, plotted against the substrate concentration, and fit to a standard linear equation as well as the following equation, which describes the binding of substrates measured by a fluorescence change, where F is the fluorescence intensity, [L] T is the total ligand concentration, F E is the fluorescence intensity at saturation, F S is the starting fluorescence signal, and K d is the dissociation constant of the substrate of interest (21). Equilibrium Binding Studies-Equilibrium dialysis was performed using a modification of the method previously described by Fersht (19). Briefly, 40 M tyrosyl-tRNA synthetase in Buffer E (150 mM Tris, pH 7.5, 150 mM KCl, 20 mM ␤-mercaptoethanol, 10 mM MgCl 2 ) was placed in one chamber (chamber A) of each cell in an 8-cell equilibrium dialyzer (Hoefer). The other chamber (chamber B) of each cell contained concentrations of L-[ 14 C]tyrosine ranging from 2 to 200 M in Buffer E. A dialysis membrane with a molecular weight cut-off of 12,000 -14,000 separated the chambers. After overnight dialysis, the L-[ 14 C]tyrosine concentrations in each chamber were determined by removing 40-l aliquots, adding each aliquot to 5 ml of Cytoscint scintillation mixture, and counting in a Beckman LS 6500 scintillation counter. The concentration of tyrosine in each chamber was calculated from the specific activity of the stock L-[ 14 C]tyrosine (5 l of the 300 M stock L-[ 14 C]tyrosine was counted to determine the specific activity). The concentrations of enzyme-bound and free tyrosine were calculated by subtracting the tyrosine concentration in chamber B ([Free]) from that in chamber A ([Bound] ϩ [Free]). The data were analyzed by both nonlinear and linear curve fitting using the following equations, where ␣ ϭ [Bound Tyrosine]/[E] T , K d is the dissociation constant for tyrosine, n is the number of binding sites, and [E] T is the total enzyme concentration (21,22). Kinetic Analyses-All kinetic analyses were performed in Buffer D at 25°C unless otherwise indicated. Stopped-flow fluorescence studies were used to monitor the tyrosine and ATP dependence of tyrosine activation and the pyrophosphate dependence of the reverse reaction in the pre-steady-state (11). 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 TyrRS⅐Tyr-AMP complex and the increase in fluorescence associated with the reverse reaction ( ex ϭ 295 nm, em Ͼ 320 nm). In the tyrosine and ATP dependence experiments, one syringe contained the enzyme (0.  (11). All kinetic data were fit to a single exponential floating end point equation using the Kaleidagraph and Applied Photophysics stopped-flow software packages to determine the observed rate constants (k obs ). The Kaleidagraph software was used to plot k obs versus the substrate concentrations and to fit these plots to the following hyperbolic function, where k 3 is the forward rate constant for the formation of tyrosyladenylate, [S] T is the total substrate concentration, and K d is the dissociation constant of the substrate of interest (21). For the pyrophosphate dependence, it was not possible to reach saturating pyrophosphate concentrations, preventing k Ϫ3 from being determined independently of K PPi . Instead, the reverse reaction was monitored under conditions where K PPi Ͼ Ͼ [PP i ], and k Ϫ3 /K PPi was determined by fitting the data to the resulting linear approximation of a hyperbolic function analogous to Equation 3. Scheme I defines the relevant rate and dissociation constants for the tyrosine activation reaction. Calculation of Relative Free Energy Levels-The relative free energies for each state along the reaction pathway were calculated from the rate and dissociation constants using the following equations, assuming standard states of 1 M for ATP, tyrosine, and pyrophosphate, which was used to calculate the Gibbs activation energy.

Comparison of the Human and B. stearothermophilus
Tyrosyl-tRNA Synthetase Amino Acid Sequences-In B. stearothermophilus tyrosyl-tRNA synthetase, site-directed mutagenesis and pre-steady state kinetic analyses have been used to identify 18 amino acids that help catalyze the formation of the tyrosyl-adenylate intermediate (reviewed in Refs. 12 and 13). Primary sequence alignment of the Rossmann-fold domains for the human and B. stearothermophilus tyrosyl-tRNA synthetases indicates that four of these 18 active site residues (Cys-35, His-48, Thr-51, and Lys-233) are not conserved in the human enzyme. In the B. stearothermophilus enzyme, three of these four amino acids, Cys-35, His-48, and Thr-51, form hydrogen bonds with the ribose ring of the ATP substrate, while the fourth amino acid, Lys-233, forms a hydrogen bond with the pyrophosphate moiety of ATP on formation of the transition state complex (1-4, 6, 14, 23, 24). In human tyrosyl-tRNA synthetase, Cys-35, His-48, Thr-51, and Lys-233 are replaced by Trp-40, Tyr-52, Pro-55, and Ser-225, respectively ( Fig. 1).
Formation of the Enzyme-bound Tyrosyl-Adenylate Intermediate Results in a Decrease in the Relative Fluorescence Emission of the Human Tyrosyl-tRNA Synthetase Above 320 nm-In B. stearothermophilus tyrosyl-tRNA synthetase, formation of the enzyme-bound tyrosyl-adenylate intermediate is associated with a decrease in the intrinsic fluorescence emission of the enzyme above 320 nm (11). To determine whether a similar fluorescence change also occurs in the human enzyme, the recombinant human tyrosyl-tRNA synthetase containing an amino-terminal His-tag/S-tag was purified and the fluorescence emission spectra of the enzyme in the presence of tyrosine, MgATP, and tyrosine ϩ MgATP were monitored (17, 25) (Fig. 2). The steady state emission spectra were determined under irreversible reaction conditions due to the presence of either pyrophosphatase (Fig. 2, panels A and B) or apyrase (an enzyme from potato that has both ATPase and ADPase activity) (Fig. 2, panel C). As a result, there is no equilibrium between the TyrRS⅐Tyr⅐ATP and TyrRS⅐Tyr-AMP⅐PP i complexes. In the absence of substrates, human tyrosyl-tRNA synthetase exhibits a relative fluorescence emission maximum at 350 nm when excited at 295 nm (Fig. 2, panels A and B). The addition of 200 M tyrosine to the human tyrosyl-tRNA synthetase causes the relative fluorescence emission spectrum to be blue-shifted by 13 (Ϯ1) nm, resulting in an 8% decrease in the fluorescence emission of the enzyme above 320 nm. This decrease is not observed when alanine is used in place of tyrosine (data not shown). Inner-filter effects due to the presence of tyrosine are negligible (Fig. 2, panel A). The addition of 10 mM MgATP to the human tyrosyl-tRNA synthetase results in a 25% decrease in the total fluorescence emission of the enzyme (Fig. 2, panel B). This observed decrease is due to inner-filter effects (discussed below). The addition of 200 M tyrosine and 10 mM MgATP together produces a 3 (Ϯ1) nm enhancement of the blue-shift observed in the presence of tyrosine alone. After correcting for the inner-filter effect of MgATP, this enhanced blue-shift results in an additional 8% decrease in the relative fluorescence of the enzyme above 320 nm. These changes in the emission spectrum of the human tyrosyl-tRNA synthetase are similar to fluorescence changes previously observed with the B. stearothermophilus enzyme on formation of the TyrRS⅐Tyr-AMP complex (11). The preformed human TyrRS⅐Tyr-AMP intermediate exhibits a relative fluorescence emission maximum at 337 nm (Fig. 2, panel C). When 1 mM tetrasodium pyrophosphate is added to this complex, the relative fluorescence emission spectrum is red-shifted by 15 (Ϯ4) nm, resulting in an increase in the relative fluorescence emission above 320 nm. This is the exact opposite of the fluorescence shift observed when human tyrosyl-tRNA synthetase is incubated with tyrosine and MgATP, and is consistent with the hypothesis that the increase in fluorescence on addition of pyrophosphate is due to the conversion of the TyrRS⅐Tyr-AMP intermediate to TyrRS ϩ Tyr ϩ ATP.
To verify that the decrease in the relative fluorescence of human tyrosyl-tRNA synthetase is due to formation of the TyrRS⅐Tyr-AMP complex, titration experiments were performed in the presence of pyrophosphatase (Fig. 3). In these experiments, the fluorescence emission of the enzyme was monitored between 320 and 400 nm. In Fig. 3, panel A, human tyrosyl-tRNA synthetase was preincubated in either the absence or presence of saturating concentrations of MgATP (10 mM). In the absence of MgATP, the addition of tyrosine resulted in an equilibrium between the free enzyme and the TyrRS⅐Tyr complex. A small decrease (8%) in the relative fluorescence emission was observed. This titration displays saturation binding, giving a dissociation constant for tyrosine (K Tyr ) of 25 (Ϯ10) M. This is in excellent agreement with the value for K Tyr obtained from equilibrium dialysis, 34 (Ϯ8) M, and is consistent with the fluorescence decrease corresponding to tyrosine binding. In the presence of 10 mM MgATP, the addition of tyrosine resulted in a much more significant (16%) decrease in the relative fluorescence emission of the enzyme. In this case, there was no equilibrium between TyrRS⅐MgATP ϩ Tyr and TyrRS⅐Tyr-AMP⅐PP i , since the addition of pyrophosphate makes the forward reaction essentially irreversible. In addition, the maximum concentration of pyrophosphate produced in the experiment, 4 M, is well below its dissociation constant (K PPi Ͼ 1 mM), preventing accumulation of the TyrRS⅐Tyr-AMP⅐PP i intermediate. Since tyrosine is irreversibly converted to tyrosyl-adenylate under these conditions, there is a linear relationship between the amount of tyrosine added and the observed decrease in fluorescence. When all of the free enzyme had been converted to the TyrRS⅐Tyr-AMP complex, no additional decrease in fluorescence was observed (Fig. 3, panel A). This is consistent with the hypothesis that the relative fluorescence decrease is due to changes in the intrinsic fluorescence of the enzyme upon the formation of the TyrRS⅐Tyr-AMP complex and not due to inner-filter effects. These results are similar to the results of titration experiments done with the B. stearothermophilus tyrosyl-tRNA synthetase (Fig. 3, panel A, inset). In contrast, when MgATP is added to human tyrosyl-tRNA synthetase in the presence of saturating concentrations of tyrosine (200 M), the relative fluorescence does not plateau once the MgATP-binding site is saturated, but instead continues to decrease as the concentration of MgATP increases (Fig. 3,  Panel B). In these experiments, the initial concentration of MgATP used was 0.4 mM, which is 100-fold larger that the enzyme concentration in the experiment. As a result, even at the lowest concentration of MgATP used, the enzyme is saturated. For this reason, the MgATP titration data were fit to a single linear equation. The linear relationship between the concentration of MgATP and the relative fluorescence emission indicates that the decrease in fluorescence upon addition of MgATP is due to inner-filter effects.
A stopped-flow reaction analyzer was used to determine whether the decrease in relative fluorescence, observed upon mixing tyrosine and MgATP with the enzyme, and the increase in relative fluorescence, observed upon mixing the pre-formed enzyme-bound tyrosyl-adenylate complex with tetrasodium pyrophosphate, could be monitored over time. These experiments were performed in the presence of pyrophosphatase. When MgATP is mixed with human tyrosyl-tRNA synthetase that has been pre-incubated with tyrosine, a rapid single exponential decrease in relative fluorescence is observed (Fig. 4, panel  A). When tyrosine is mixed with enzyme that has been preincubated with MgATP, a similar rapid single exponential decrease in relative fluorescence is observed, which has the same k obs and amplitude as the decrease observed when MgATP is added to the enzyme preincubated with tyrosine. To determine whether the observed fluorescence change corresponds to the initial binding of the substrates to the enzyme, the enzyme was mixed with either tyrosine or MgATP alone. Although a de- crease in the relative fluorescence is observed when either tyrosine or MgATP alone are mixed with the enzyme in the stopped-flow instrument, the k obs for the fluorescence changes are too slow to correspond to the initial binding of these substrates to the active site of the enzyme (data not shown). In addition, the magnitude of the decrease observed in the presence of both substrates is more that twice the magnitude of the decrease observed in the presence of either tyrosine or MgATP alone, and the rate of this single decrease is more than 50 times faster. The ability to fit the rapid decrease to a single exponential equation is consistent with a rapid equilibrium assumption for the initial binding of tyrosine and MgATP to the active site of the free enzyme. One possible explanation for these slow fluorescence changes observed upon addition of tyrosine or MgATP alone is that they correspond to the binding of either tyrosine or MgATP to the inactive subunit of tyrosyl-tRNA synthetase. For the reverse reaction, a rapid single exponential increase in relative fluorescence was observed when pyrophosphate was mixed with the pre-formed TyrRS⅐Tyr-AMP complex (Fig. 4, Panel B). This observed increase in relative fluorescence mirrors the decrease observed when free enzyme is mixed with both tyrosine and MgATP. In these stopped-flow experiments, the equilibrium strongly favors formation of the free enzyme, as the total concentrations of tyrosine and MgATP released from the TyrRS⅐Tyr-AMP complex are well below their dissociation constants ([Tyr] ϭ [MgATP] ϭ 0.5 M versus K Tyr ϭ 34 M, K ATP Ј ϭ 4.0 mM). Since the equilibrium strongly favors formation of the free enzyme under these conditions, it is not necessary to add apyrase to convert ATP to AMP ϩ 2P i to ensure that the reaction is irreversible. The ability to fit the increase in relative fluorescence to a single exponential equation is consistent with a rapid equilibrium assumption for the binding of tetrasodium pyrophosphate to the TyrRS⅐Tyr-AMP complex. The observations that: 1) the observed relative fluorescence of the human tyrosyl-tRNA synthetase decreases when both tyrosine and MgATP are added and 2) the observed relative fluorescence of the TyrRS⅐Tyr-AMP complex increases when pyrophosphate is added, are consistent with the hypothesis that changes in the intrinsic fluorescence of tyrosyl-tRNA synthetase are associated with the formation and breakdown of the TyrRS⅐Tyr-AMP complex.

Catalysis of Tyrosine Activation by Human Tyrosyl-tRNA
Synthetase Is Potassium Dependent-Catalysis of tRNA Tyr aminoacylation by tyrosyl-tRNA synthetases from porcine thyroid glands, Saccharomyces cerevisiae, Drosophila, and wheat germ has previously been shown to require potassium (26 -29).
To determine whether potassium is required for tyrosine activation by the human tyrosyl-tRNA synthetase, the pre-steady state kinetics for formation of the TyrRS⅐Tyr-AMP intermediate were monitored in the absence and presence of 150 mM KCl. Potassium chloride was used at this concentration because it is similar to the intracellular concentration of potassium in a typical eukaryotic cell (30). As shown in Fig. 5, panel A, formation of the tyrosyl-adenylate intermediate is stimulated by the presence of 150 mM KCl. Fitting plots of k obs versus tyrosine concentration to Equation 3 indicated that the addition of 150 mM KCl increases the forward rate constant for the reaction (k 3 ) by ϳ260-fold under the conditions used here (Fig. 5, panel A). The dissociation constant for MgATP (K ATP Ј ) is also decreased in the presence of 150 mM KCl (data not shown). These effects were not observed when 150 mM NaCl was used in place of KCl (data not shown). Formation of the tyrosyl-adenylate intermediate at various concentrations of KCl in the presence of saturating concentrations of tyrosine and MgATP indicated that the dissociation constant for K ϩ is 32 (Ϯ2) mM (Fig. 5, panel B). Calculation of the activation energy for tyrosine activation in the absence and presence of KCl indicated that potassium decreases the Gibbs activation energy from 18.5 to 15.2 kcal/mol.

The Human Tyrosyl-tRNA Synthetase Displays Half-of-thesites
Reactivity-Previous studies indicate that B. stearothermophilus tyrosyl-tRNA synthetase is a homodimeric enzyme that displays half-of-the-sites reactivity with respect to tyrosine binding and tyrosyl-adenylate formation (9 -11). The human tyrosyl-tRNA synthetase is also a homodimer (31). Equilibrium dialysis was used to determine whether the human tyrosyl-tRNA synthetase displays half-of-the-sites reactivity with respect to tyrosine binding. Specifically, the equilibrium dialysis data were fit to Equation 2 to calculate both the dissociation constant for tyrosine (K Tyr ) and the number of tyrosine binding sites per tyrosyl-tRNA synthetase dimer. The dissociation constant for tyrosine for the human tyrosyl-tRNA synthetase as determined by equilibrium dialysis is 34 (Ϯ8) M (Fig. 6, panel A, Table I). Equilibrium dialysis also indicates that the human tyrosyl-tRNA synthetase binds 1.16 (Ϯ0.03) tyrosine molecules per enzyme dimer (i.e. it displays half-ofthe-sites reactivity). This is consistent with comparison of the enzyme concentration determined by A 280 measurements and by active site titration, which indicate that the enzyme displays half-of-the-sites reactivity with respect to tyrosyl-adenylate formation (data not shown).
Comparison of Tyrosine Activation Catalyzed by the Human and B. stearothermophilus Tyrosyl-tRNA Synthetases-To determine whether human tyrosyl-tRNA synthetase is less active with respect to tyrosine activation than the B. stearothermophilus enzyme, the relative rate and dissociation constants for tyrosine activation were determined for the human tyrosyl-tRNA synthetase and compared with those of the B. stearothermophilus enzyme. Unless otherwise indicated, the concentra- tion of KCl was 150 mM in these experiments. Equilibrium dialysis was used to determine K Tyr (Fig. 6, panel A and Table  I) and pre-steady state kinetic methods were used to determine the forward rate constant, k 3 , and the dissociation constant for MgATP, K ATP Ј (Table I). Similarly, pre-steady state kinetic methods were used to monitor the reverse reaction. The kinetic analyses of the reverse reaction were complicated by the precipitation of pyrophosphate in the presence of 150 mM KCl when the concentration of pyrophosphate exceeded 1 mM. To minimize the effect that KCl has on the solubility of the pyrophosphate substrate, the pre-steady state kinetics for the reverse reaction were determined in the presence of 75 mM KCl. Even at this lower KCl concentration, however, we were unable to prevent the precipitation of pyrophosphate above 1 mM. As a result, it was not possible to monitor the reverse reaction under conditions where the concentration of pyrophosphate saturates the enzyme-binding sites. This prevents the reverse rate constant (k Ϫ3 ) and dissociation constant for pyrophosphate (K PPi ) from being calculated independently of each other. However, it is still possible to calculate the specificity constant (k Ϫ3 /K PPi ) for the reverse reaction using conditions where K PPi Ͼ Ͼ [PP i ] and fitting the data to the resulting linear approximation of a hyperbolic function analogous to Equation 3 (Fig. 6, panel B, Table I).
Comparison of the dissociation constant for the human tyrosyl-tRNA synthetase with previously published data for the B. stearothermophilus enzyme indicates that the tyrosine substrate binds to the human tyrosyl-tRNA synthetase with an affinity 3-fold lower than that by which it binds to the B. stearothermophilus enzyme (Table I). In contrast, both the dissociation constant for MgATP and the rate constant for formation of the tyrosyl-adenylate intermediate are similar for the human and B. stearothermophilus enzymes. There is a 2-fold difference in the specificity constants for the reverse reaction.
The recombinant human tyrosyl-tRNA synthetase used in these studies contains an amino-terminal His 6 -tag/S-tag sequence followed by an enterokinase proteolytic site immediately preceding the coding sequence for the human tyrosyl-tRNA synthetase. To determine whether the presence of the His 6 -tag/S-tag sequence affects the activity of the human tyrosyl-tRNA synthetase, this sequence was removed by enterokinase digestion and the non-tagged enzyme was assayed for activity. Although analysis of the enterokinase reaction products on SDS-PAGE indicates that there is a non-canonical enterokinase site within the human tyrosyl-tRNA synthetase sequence, it was possible to optimize the reaction conditions to yield primarily full-length human tyrosyl-tRNA synthetase (data not shown). Comparison of the rate and dissociation constants for the human tyrosyl-tRNA synthetase with and without the amino-terminal His 6 -tag/S-tag indicates that the presence of this amino-terminal sequence has little effect on the kinetics of the recombinant enzyme (Table I).
Calculation of the Free Energy Profile for the Human Tyrosyl-tRNA Synthetase-To determine whether any of the bound complexes along the reaction pathway are less stable in the human tyrosyl-tRNA synthetase than in the B. stearothermophilus enzyme, the Gibbs free energy values for each bound state in the reaction pathway were calculated relative to the free energy of the unliganded enzyme (Fig. 7). Values shown for the B. stearothermophilus enzyme are taken from previously published data (3). Due to the inability to determine k Ϫ3 independently of K PPi , it is not possible to calculate the relative free energy for the human TyrRS⅐Tyr-AMP⅐PP i intermediate complex and therefore this complex is omitted in Fig. 7. Although the weaker binding of the tyrosine substrate results in a slight (0.4 kcal/mol) destabilization of the transition state complex relative to the free energy of unliganded enzyme for the human tyrosyl-tRNA synthetase, the Gibbs activation energies are identical for the two enzymes (15.2 kcal/mol).

DISCUSSION
Catalysis of tyrosine activation by B. stearothermophilus tyrosyl-tRNA synthetase is associated with a change in the intrinsic fluorescence of the enzyme (11). In this paper, we demonstrate that changes in the intrinsic fluorescence of human tyrosyl-tRNA synthetase can be used to monitor the tyrosine activation step of the aminoacylation reaction. A number of lines of evidence support this statement. First, analysis of the steady-state fluorescence emission spectrum of human tyrosyl-tRNA synthetase indicates that the addition of both the tyrosine and MgATP substrates to the enzyme causes a decrease in its relative fluorescence emission above 320 nm. This fluorescence decrease is similar to the decrease in intrinsic fluorescence observed in the B. stearothermophilus enzyme on addition of tyrosine and MgATP. In particular, the observation that the fluorescence decrease for the human tyrosyl-tRNA synthetase plateaus at saturating concentrations of tyrosine and MgATP indicates that the change in fluorescence results from the formation of tyrosyl-adenylate, not from inner-filter effects. While the initial binding of tyrosine to the human tyrosyl-tRNA synthetase contributes to the blue-shift observed in the steady-state fluorescence emission spectrum, subsequent addition of MgATP results in an additional blue-shift in the spectrum. The dissociation constant for tyrosine calculated from the fluorescence titration experiments is in excellent agreement with that calculated from equilibrium dialysis experiments and is consistent with the hypothesis that the initial blue-shift corresponds to tyrosine binding. The additional blueshift can be ascribed to the conversion of the TyrRS⅐Tyr⅐ATP complex to the TyrRS⅐Tyr-AMP intermediate. Second, the addition of pyrophosphate to the TyrRS⅐Tyr-AMP intermediate causes an increase in the steady-state fluorescence intensity of the sample, presumably due to the conversion of TyrRS⅐Tyr-AMP ϩ PP i to TyrRS ϩ Tyr ϩ ATP (Scheme I). Supporting this interpretation is the observation that the red-shift in the steady-state fluorescence emission spectrum observed on addition of pyrophosphate to the TyrRS⅐Tyr-AMP intermediate is similar in magnitude to the blue-shift observed on addition of tyrosine and MgATP to the free enzyme. Third, when the enzyme is mixed with tyrosine and MgATP in a stopped-flow reaction analyzer, a time-dependent exponential decrease in the relative fluorescence is observed. A plot of k obs versus substrate concentration indicates that this time-dependent fluorescence decrease displays saturation kinetics with respect to tyrosine and MgATP concentrations. The observation that the fluorescence change, independent of the order of substrate addition, follows a single exponential equation with no slow reactions observed at extended reaction times is consistent with both a rapid equilibrium assumption for the binding of tyrosine and MgATP and the hypothesis that the observed decrease in fluorescence corresponds to formation of the TyrRS⅐Tyr-AMP intermediate. The absence of the slower decrease at extended observation times suggests that the fluorescence decreases observed in the presence of either tyrosine or MgATP alone are probably due to binding to the second subunit, since formation of Tyr-AMP at the active site of one subunit would inhibit the binding of MgATP or tyrosine to the other subunit due to half-of-the-sites reactivity. Fourth, when the TyrRS⅐Tyr-AMP intermediate is mixed with pyrophosphate in a stopped-flow reaction analyzer, a time-dependent exponential increase in the relative fluorescence of the enzyme is observed. The observations that this fluorescence increase can be fit to a single order exponential equation, and that it mirrors the fluorescence decrease observed when the free enzyme is mixed with both tyrosine and MgATP, further support the hypothesis that the changes in fluorescence are associated with the formation and breakdown of the TyrRS⅐Tyr-AMP complex. All of the above observations are consistent with previous observations on the B. stearothermophilus enzyme and suggest that, like the B. stearothermophilus enzyme, the human tyrosyl-tRNA synthetase undergoes a conformational change on formation of the tyrosyl-adenylate intermediate (11). The observation that rate and dissociation constants determined from stopped-flow fluorescence experiments are very similar for both the human and B. stearothermophilus tyrosyl-tRNA synthetases further supports the hypothesis that, like the B. stearothermophilus en-zyme the fluorescence change observed for the human enzyme is associated with the chemical step of the tyrosine activation reaction. It is likely that this conformational change is a common feature in the catalytic mechanism of all tyrosyl-tRNA synthetases.
The observation that, unlike the bacterial enzyme, the human tyrosyl-tRNA synthetase requires potassium for tyrosine activation, suggests that the potassium dependence may be specific to eukaryotes. Previous studies showing that potassium is required for the catalysis of tRNA Tyr aminoacylation by other eukaryotic tyrosyl-tRNA synthetases further support this hypothesis (26 -29). It is not clear how potassium activates eukaryotic tyrosyl-tRNA synthetases. One possibility is that potassium is required for dimerization of the enzyme subunits. Alternatively, potassium may be required for stabilization of the tertiary structure of eukaryotic tyrosyl-tRNA synthetases. A third possibility is that the binding of potassium induces a conformational change that organizes or brings additional functional groups into the active site of the enzyme. Last, potassium may bind in the active site of the enzyme and interact directly with one or more of the substrates during formation of the transition state complex. The specific role that potassium cations play in the activation of tyrosyl-tRNA synthetase is currently being investigated.
The aminoacyl-tRNA synthetases have drawn interest as potential targets for antibiotics (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45). In particular, Stefanska et al. (37) have recently isolated a series of related competitive inhibitors of tyrosyl-tRNA synthetase that bind 40,000fold more tightly to Staphyloccocus aureus tyrosyl-tRNA synthetase than it does to the S. cerevisiae enzyme (39 -41, 46 -50). The selectivity of these inhibitors suggests that differences exist between the active sites of bacterial and eukaryotic tyrosyl-tRNA synthetases. Identifying these differences will facilitate the development of additional compounds that selectively inhibit bacterial tyrosyl-tRNA synthetase. Comparison of bacterial, archaeal, and eukaryotic tyrosyl-tRNA synthetase sequences indicates that while the majority of active site residues are conserved among all three domains of life, several amino acids that stabilize the transition state for tyrosine activation in the B. stearothermophilus enzyme are not conserved in the archaeal and eukaryotic homologues. Cys-35 stabilizes the transition state in B. stearothermophilus tyrosyl-tRNA synthetase by 1.2 kcal/mol through interactions with the 3Ј hydroxyl of ATP. Despite its role in stabilizing the transition state in the B. stearothermophilus enzyme, replacement of Cys-35 by a tryptophan in the human enzyme is plausible, as this residue is conserved in only 10 of 50 bacterial tyrosyl-tRNA synthetases examined. In contrast, the lack of conservation of His-48 (the second histidine in the HIGH sequence motif, conserved in 43 of 50 bacterial tyrosyl-tRNA synthetase sequences) and Lys-233 (the second lysine in the KMSKS sequence motif, conserved in all 50 of the bacterial tyrosyl-tRNA synthetase sequences analyzed) is surprising. In B. stearothermophilus tyrosyl-tRNA synthetase, His-48 forms a hydrogen bond with the 4Ј-oxygen atom in the ribose ring of ATP. This residue is replaced by a tyrosine in the human enzyme. It is possible that this tyrosine forms a hydrogen bond with the 4Ј-oxygen atom in the ribose ring of ATP. In addition, the phenyl ring of the tyrosine residue may interact with the ribose ring of ATP providing additional binding energy for catalysis. The replacement of Lys-233 in B. stearothermophilus tyrosyl-tRNA synthetase by a serine residue in the human enzyme is more difficult to rationalize. In the B. stearothermophilus enzyme, Lys-233 interacts with the pyrophosphate moiety of ATP and stabilizes the transition state for tyrosine activation by 3.0 kcal/mol (4, 6). In addition, Lys-233 is the most highly con- served amino acid in the class I aminoacyl-tRNA synthetase family (51), suggesting that its replacement by serine in eukaryotic tyrosyl-tRNA synthetases is a relatively recent evolutionary event. The observation that the B. stearothermophilus and human tyrosyl-tRNA synthetases exhibit nearly identical activation energies for the first step of the tRNA Tyr aminoacylation reaction implies that the human enzyme has somehow compensated for the absence of Lys-233. One possibility is that Lys-233 is replaced by a nearby basic residue in the human enzyme. In human tyrosyl-tRNA synthetase, there is a lysine residue located five amino acids past the KMSKS sequence motif. This lysine is present in all of the known eukaryotic tyrosyl-tRNA synthetase sequences in which Lys-233 is not conserved, making it a candidate for functionally replacing Lys-233. Alternatively, the human tyrosyl-tRNA synthetase may compensate for the absence of both Cys-35 and Lys-233 through interactions with the transition state complex outside of the 3Ј hydroxyl and pyrophosphate groups of ATP. One candidate for this role is the replacement of an active site threonine in B. stearothermophilus tyrosyl-tRNA synthetase (Thr-51) by a proline in the human enzyme (Pro-55). In B. stearothermophilus tyrosyl-tRNA synthetase, the replacement of Thr-51 with a proline residue stabilizes the transition state by 2.2 kcal/mol (24). Although the replacement of Thr-51 with proline in the human enzyme does not functionally compensate for the absence of either Cys-35 or Lys-233 in the active site, it may compensate energetically for some of the lost binding energy associated with these amino acids. Last, given the potassium dependence of the human tyrosyl-tRNA synthetase, it is likely that this cation plays a role in compensating for the absence of either Cys-35 or Lys-233 in the active site of the human enzyme.
In this paper, we have demonstrated that changes in intrinsic fluorescence can be used to monitor the pre-steady state kinetics of human tyrosyl-tRNA synthetase. In addition, we have shown that the human tyrosyl-tRNA synthetase requires potassium to stabilize the transition state for tyrosine activation. We have further shown that the human and B. stearothermophilus enzymes display similar kinetics for the formation of the tyrosyl-adenylate intermediate, despite differences in the catalytic residues present in their active sites. The results of these investigations suggest that differences exist between bacterial and human tyrosyl-tRNA synthetases that can be exploited in the design of antibiotics.