Correlating amino acid conservation with function in tyrosyl-tRNA synthetase1

doi:10.1006/jmbi.2000.4125 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 303, 287±298

Correlating Amino Acid Conservation with Function in Tyrosyl-tRNA Synthetase Yu Xin1, Weidong Li1, Donard S. Dwyer2 and Eric A. First1* 1

Department of Biochemistry and Molecular Biology 2

Department of Pharmacology and Psychiatry, Louisiana State University Health Sciences Center, Shreveport LA 71130, USA

Sequence comparisons have been combined with mutational and kinetic analyses to elucidate how the catalytic mechanism of Bacillus stearothermophilus tyrosyl-tRNA synthetase evolved. Catalysis of tRNATyr aminoacylation by tyrosyl-tRNA synthetase involves two steps: activation of the tyrosine substrate by ATP to form an enzyme-bound tyrosyl-adenylate intermediate, and transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNATyr. Previous investigations indicate that the class I conserved KMSKS motif is involved in only the ®rst step of the reaction (i.e. tyrosine activation). Here, we demonstrate that the class I conserved HIGH motif also is involved only in the tyrosine activation step. In contrast, one amino acid that is conserved in a subset of the class I aminoacyl-tRNA synthetases, Thr40, and two amino acids that are present only in tyrosyl-tRNA synthetases, Lys82 and Arg86, stabilize the transition states for both steps of the tRNA aminoacylation reaction. These results imply that stabilization of the transition state for the ®rst step of the reaction by the class I aminoacyl-tRNA synthetases preceded stabilization of the transition state for the second step of the reaction. This is consistent with the hypothesis that the ability of aminoacyl-tRNA synthetases to catalyze the activation of amino acids with ATP preceded their ability to catalyze attachment of the amino acid to the 30 end of tRNA. We propose that the primordial aminoacyl-tRNA synthetases replaced a ribozyme whose function was to promote the reaction of amino acids and other small molecules with ATP. # 2000 Academic Press

*Corresponding author

Keywords: tyrosyl-tRNA synthetase; aminoacylation; pre-steady state kinetics; evolution; RNA world

Introduction The aminoacyl-tRNA synthetases (AARS) can be divided into two distinct classes based on structural and sequence similarities (Cusack et al., 1990; Eriani et al., 1990). Both classes catalyze the aminoacylation of tRNA by a two-step mechanism in which amino acid activation (equation (1)) is followed by transfer of the amino acid to the 30 end of tRNA (equation (2)): AARS ‡ AA ‡ ATP „ AARS
AA-AMP ‡ PPi …1† Abbreviations used: AA, amino acid(s); AARS, aminoacyl-tRNA synthetase(s); PPi, pyrophosphate; -, covalent bond;
, non-covalent bond; AARS
AA-AMP, enzyme-bound aminoacyl-adenylate; AA-tRNAAA, aminoacyl-tRNA; TyrRS, tyrosyl-tRNA synthetase. E-mail address of the corresponding author: e®[email protected] 0022-2836/00/020287±12 $35.00/0

AARS
AA-AMP ‡ tRNAAA „ AARS ‡ AMP ‡ AA-tRNAAA

…2†

Class I aminoacyl-tRNA synthetases are characterized by their Rossmann fold domain (Rossmann et al., 1974), which contains the binding sites for the amino acid and ATP substrates, as well as the 30 end of their cognate tRNA. In addition, two sequence motifs, HIGH and KMSKS, are highly conserved among the class I aminoacyl-tRNA synthetases (Webster et al., 1984; Hountondji et al., 1986). These two motifs have been shown to stabilize the transition state for the ®rst step of the aminoacylation reaction (Wells & Fersht, 1986; Borgford et al., 1987; Leatherbarrow & Fersht, 1987; First & Fersht, 1993a,b,c, 1995). In contrast, in the class II aminoacyl-tRNA synthetases, the amino acid and ATP substrates bind to a seven-stranded antiparallel b-sheet that is # 2000 Academic Press

288

Evolution of Catalysis in Tyrosyl-tRNA Synthetase

packed between two long a-helices and is unrelated to the Rossmann fold domain (Cusack et al., 1990; Eriani et al., 1990). Tyrosyl-tRNA synthetase belongs to the class I aminoacyl-tRNA synthetase family (Eriani et al., 1990). Bacillus stearothermophilus tyrosyl-tRNA synthetase is a homodimer that displays ``half-ofthe-sites'' reactivity, with the binding of tyrosine to one subunit inactivating the other subunit (Jakes & Fersht, 1975). The anticodon loop of tRNATyr binds to the carboxyl-terminal domain of this inactive subunit, with the 30 end of tRNATyr binding to the Rossmann fold domain of the other subunit (Waye et al., 1983; Carter et al., 1986). Previous investigations have shown that His45 and His48 in the HIGH motif, Lys230, Lys233, and Thr234 in the KMSKS motif, and Thr40, Lys82, and Arg86 stabilize the transition state for tyrosyl-adenylate formation (Leatherbarrow et al., 1985; Lowe et al., 1985; Jones et al., 1986; Leatherbarrow & Fersht, 1987; Fersht et al., 1988; First & Fersht, 1993a,b,c, 1995). With the exception of His48, which forms a hydrogen bond with the oxygen atom in the ribose ring of ATP, all of these residues are hydrogen bonded to the pyrophosphate moiety of ATP in the transition state for the ®rst step of the reaction. We have recently shown that although the conserved KMSKS motif has a modest effect on the initial binding of tRNATyr, it does not alter the activation energy for the second step of the reaction (Xin et al., 2000a). In this study, we test the hypothesis that Thr40, His45, His48, Lys82, and Arg86 play catalytic roles for the second step of the aminoacylation reaction. Comparison of the degree of sequence conservation for these amino acids with their role in the catalytic mechanism of tyrosyl-tRNA synthetase gives a picture of how the catalytic mechanism of tyrosyl-tRNA synthetase has evolved. The data presented here are consistent with the hypothesis that catalysis of the amino acid activation step preceded catalysis of the amino acid transfer step by the aminoacyl-tRNA synthetases, and suggests that the original function of the aminoacyl-tRNA synthetases may have been to supply a primordial ribozyme-based translation system with activated amino acids for use as substrates in protein synthesis.

Results The class I conserved HIGH sequence motif does not participate in the second step of the aminoacylation reaction To study the role that the HIGH sequence motif plays in the second step of the aminoacylation reaction, His45 and His48 were replaced by alanine (designated H45A and H48A, respectively). Presteady-state kinetic analyses were performed using a stopped-¯ow ¯uorescence assay, which monitors the increase in the intrinsic ¯uorescence of tyrosyltRNA synthetase upon formation of the transition state (E
[Tyr-tRNATyr
AMP]{) complex (Avis et al.,1993). No ¯uorescence changes were observed upon the initial binding of in vitro transcribed tRNATyr. This is consistent with previous observations by Avis et al. (1993). For each variant, the dependence of the initial rate on the concentration of tRNATyr was determined. Binding and rate constants were calculated by ®tting the substratedependence of the initial rate on the tRNATyr concentration to the Michaelis-Menten equation. Binding and rate constants for the H45A and H48A variants are similar to those of the wild-type tyrosyl-tRNA synthetase and are summarized in Table 1. Threonine 40, lysine 82, and arginine 86 stabilize the transition state for the second step of the reaction In the transition state for the ®rst step of the reaction, Thr40, Lys82, and Arg86 form hydrogen bonds with the pyrophosphate moiety of ATP (Leatherbarrow et al., 1985; Leatherbarrow & Fersht, 1987; Fersht et al., 1988). To determine whether these amino acids stabilize the transition state for the second step of the aminoacylation reaction, alanine variants at each of these positions were analyzed using pre-steady-state kinetics. Typical Michaelis-Menten plots for the T40A, K82A, and R86A variants are shown in Figure 1(a), (c) and (d). Binding and rate constants calculated for each of the variants are summarized in Table 1. The T40A, K82A, and R86A variants all show decreased values for k4, the rate constant for the formation of the transition state for the second step

Table 1. Binding and rate constants for the tyrosyl-tRNA synthetase variants Enzyme variant

KtRNA (mM)

k4 (sÿ1)

k4/KtRNA (sÿ1 mMÿ1)

Wild-type T40A H45A H48A K82A R86A

0.39  (0.03) 1.0  (0.1) 0.49  (0.02) 0.45  (0.01) 1.2  (0.1) 2.0  (0.1)

31  (1) 4.3  (0.1) 26  (1) 26  (1) 4.8  (0.1) 0.47  (0.01)

79 4.3 53 58 4.0 0.24

Experimental errors, which are determined by calculating the standard deviations for three repetitive experiments, are indicated in parentheses.

Evolution of Catalysis in Tyrosyl-tRNA Synthetase

289

Figure 1. Typical Michaelis-Menten plots for the transfer of tyrosine to in vitro transcribed B. stearothermophilus tRNATyr substrate are shown for the (a) T40A, (b) H45A, (c) K82A, and (d) R86A tyrosyl-tRNA synthetase variants. The insets are Eadie-Hoftsee transformations of the data. The concentration of the E
Tyr-AMP complex is 0.5 mM in each of these assays, as determined by Bradford assay using wild-type tyrosyl-tRNA synthetase as the standard protein (Bradford, 1976). Standard deviations from a least-squares ®t of the single exponential ¯oating end point equation for the rate curves are shown as double hatched bars. The Michaelis-Menten plot for the H48A variant (not shown) is similar to that of the wild-type enzyme.

of the reaction, indicating that they all destabilize this transition state. This is particularly apparent in the R86A variant, whose k4 value is 66-fold less than that of the wild-type enzyme. The T40A, K82A, and R86A variants display increased KtRNA values, indicating that these three residues play a role in the initial binding of tRNATyr. These data are consistent with the observation that the K82N and R86Q variants strongly reduce the rate for the transfer of tyrosine from E
Tyr-AMP to tRNATyr in the second step of the aminoacylation reaction (A. Fersht & H. Bedouelle, personal communication). The relative free energies for the binding of the tyrosyl-tRNA synthetase variants to both the E
TyrAMP
tRNATyr and E
[Tyr-tRNATyr
AMP]{ complexes were calculated from the KtRNA and k4 values

for each variant. Relative free energies for the variant enzymes are compared to those of the wild-type enzyme in the apparent free energy diagram shown in Figure 2. Neither the H45A nor the H48A variant differs signi®cantly from the wild-type enzyme in its ability to stabilize the transition state complex. In contrast, the T40A, K82A, and R86A variants destabilize both the E
Tyr-AMP
tRNATyr and E
[TyrtRNATyr
AMP]{ complexes. Relative to the wildtype enzyme, the T40A, K82A, and R86A variants destabilize the E
Tyr-AMP
tRNATyr complex by 0.56, 0.65, and 0.98 kcal/mol, respectively. The effect of these variants is much larger in the transition state, with T40A, K82A, and R86A destabilizing the E
[Tyr-tRNATyr
AMP]{ complex by 1.73, 1.76, and 3.46 kcal/mol, respectively.

290

Evolution of Catalysis in Tyrosyl-tRNA Synthetase

Figure 2. The apparent free energy changes for the T40A, H45A, H48A, K82A, and R86A variants. The free energy differences between each of the variants and the wild-type enzyme (

Gapp) are shown for each enzyme-bound state in the second step of the tRNATyr aminoacylation reaction. The free energy difference for each state in the reaction pathway is calculated by subtracting the relative free energy of the wild-type enzyme from the relative free energy for the variant enzyme (i.e.

Gapp ˆ
Gvariant ÿ
Gwild-type). Positive values of

Gapp indicate that replacement of the wild-type amino acid with alanine destabilizes the bound state. Standard deviations of three repetitive experiments are indicated by single hatched lines on the top of column bars. The reference state for calculation of the relative free energies is the E
Tyr-AMP complex.

Replacement of threonine 40, histidine 45, histidine 48, lysine 82, or arginine 86 by alanine results in differential destabilization of the transition states for the reaction To evaluate the contributions of each amino acid residue on the reaction pathway, a free energy pro®le for each of the tyrosyl-tRNA synthetase variants has been calculated (Figure 3). In the wildtype enzyme, the free energy of the transition state for the formation of the tyrosyl-adenylate intermediate (E
[Tyr-ATP]{) is identical (within experimental error) with the free energy of the transition state for the transfer of tyrosine to tRNATyr (Avis et al., 1993). In contrast, the effect of introducing an alanine residue at position 45 is to destabilize the E
[Tyr-ATP]{ complex without affecting the stability of the E
[Tyr-tRNATyr
AMP]{ complex (Figure 3(b)). The reaction pro®le for the H48A variant is similar to that of the H45A variant (data not shown). In effect, catalysis of tRNATyr aminoacylation by the H45A and H48A tyrosyl-tRNA synthetase variants has a single rate-limiting step, formation of the E
[Tyr-ATP]{ complex. This is similar to the effect that replacing K230, K233, or T234 with alanine has on the free energy pro®le of the reaction (Xin et al., 2000a). The situation is somewhat different for the T40A, K82A, and R86A variants, as the introduction of alanine at each of these positions destabilizes the transition states for both steps of the reaction. For the T40A variant (Figure 3(a)), replacing the threonine side-chain with the alanine side-chain has a much greater affect on the E
[Tyr-ATP]{ complex than it does on the E
[Tyr-tRNATyr
AMP]{ complex (T40A destabilizes the E
[Tyr-ATP]{ and E
[Tyr-tRNATyr
AMP]{ complexes by 5.05 and 1.73 kcal/mol, respectively). Thus in the T40A variant, formation

of the E
[Tyr-ATP]{ complex is the rate-limiting step for the reaction. Similarly, the K82A and R86A variants destabilize the E
[Tyr-ATP]{ complex to a greater extent than they destabilize the E
[TyrtRNATyr
AMP]{ complex (3.05 and 1.76 kcal/mol, respectively for K82A, and 5.34 and 3.46 kcal/mol, respectively for R86A). Modeling the docking of tRNAPhe to tyrosyl-tRNA synthetase The docking of tRNAPhe to tyrosyl-tRNA synthetase was performed by ®rst positioning the tRNAPhe structure such that the stem region ®t into the acceptor-binding site in the tyrosyl-tRNA synthetase dimer. The interface between these two molecules was re®ned to satisfy constraints based on the results presented in this and the accompanying paper (Xin et al., 2000b), and those of Bedouelle and his colleagues (Bedouelle & Winter, 1986; Bedouelle, 1990; Bedouelle et al., 1993). This initial model resulted in a poor ®t between the 30 terminus of tRNAPhe and the active site of tyrosyltRNA synthetase. This is presumably due to differing conformations of the 30 terminus for the crystal structure of tRNAPhe and the structure of tRNATyr when it is bound to tyrosyl-tRNA synthetase. This discrepancy is not unreasonable, as the 30 end of tRNA has been shown to adopt multiple conformations (Jack et al., 1976; Hingerty et al., 1978; Perona et al., 1993; Goldgur et al., 1997; Eiler et al., 1999; Sankaranarayanan, 1999; Silvian et al., 1999). To obtain a better ®t for the 30 terminus of tRNAPhe, nucleotides C75 and A76 were rotated about their phosphoester bonds such that A76 was positioned within a pocket formed by Thr40, Lys82, and Arg86, and the 20 hydroxyl group of A76 was in close proximity to the acylphosphate

291

Evolution of Catalysis in Tyrosyl-tRNA Synthetase

Figure 3. Free energy diagrams for the formation of each complex in the tRNATyr aminoacylation reaction. Continuous lines indicate the free energy changes during the course of the reaction for the wild-type tyrosyl-tRNA synthetase. Broken lines indicate the effects that the (a) T40A, (b) H45A, (c) K82A, and (d) R86A substitutions have on the stability of each enzyme-bound complex in the aminoacylation reaction. The reaction pro®le for wild-type tyrosyltRNA synthetase is taken from Avis et al. (1993). Values for the T40A, H45A, K82A, and R86A variants for the ®rst step of the reaction are taken from previously published reports (Leatherbarrow et al., 1985; Wells & Fersht, 1986; Leatherbarrow & Fersht, 1987; Fersht et al., 1988). The reaction pro®le for the H48A variant (not shown) is similar to that of the H45A variant.

bond in the tyrosinyl-adenylate inhibitor. The tRNAPhe in this model is rotated by approximately 25  relative to the tRNA in the model proposed by Bedouelle and colleagues (Figure 4(a) and (b); Bedouelle & Winter, 1986; Bedouelle, 1990; Bedouelle et al., 1993). One effect of this rotation is that it brings the anticodon arm of tRNAPhe within Ê of the KMSKS motif in the inactive subunit of 10 A tyrosyl-tRNA synthetase. This is consistent with previous results that indicate the KMSKS motif, which is located on a mobile loop in tyrosyl-tRNA synthetase (Fersht et al., 1988), may interact with the anticodon arm of tRNATyr (Xin et al., 2000a). Although the tRNAs are bound in slightly different orientations in the two models, the model presented here is in good agreement with the model proposed by Bedouelle and colleagues with respect to the position of adenosine 76 within the active site of the enzyme. Speci®cally, in the model preÊ away from O4 in the sented here, Thr40 is 4.8 A Ê away ribose ring of adenosine 76, Lys82 is 4 A 0 from the 3 hydroxyl group of the ribose ring, and Ê away from O5 in the ribose moiety Arg86 is 3.5 A of adenosine 76 (Figure 4(c)).

Discussion Mechanistic interpretation of the role that Thr40, Lys82, and Arg86 play in the second step of the aminoacylation reaction Bedouelle and colleagues (Bedouelle & Winter, 1986; Bedouelle, 1990; Bedouelle et al., 1993) constructed a structural model between tyrosyl-tRNA synthetase and tRNAPhe by successive cycles of prediction, mutagenesis of tyrosyl-tRNA synthetase to test their predictions, and molecular modeling. In this model, one molecule of tRNA binds per tyrosyl-tRNA synthetase dimer. The acceptor arm of tRNATyr interacts with the amino-terminal Rossmann fold domain of one tyrosyl-tRNA synthetase subunit and the anticodon arm is in a position suitable to interact with the disordered carboxyl-terminal domain in the other subunit. The 30 end of tRNATyr (i.e. C74C75A76) is elongated in order to reach the tyrosyl-adenylate intermediate and its bases are oriented towards the solvent. Positioning of the 30 terminus of tRNATyr suggests that adeno-

292

Evolution of Catalysis in Tyrosyl-tRNA Synthetase

Figure 4. A structural model for the docking of yeast tRNAPhe to B. stearothermophilus tyrosyl-tRNA synthetase. In (a) and (b), the structural model presented here for the docking of tRNAPhe to the B. stearothermophilus tyrosyl-tRNA synthetase dimer is compared with the model proposed by Bedouelle and colleagues (Bedouelle & Winter, 1986; Bedouelle, 1990; Bedouelle et al., 1993). To facilitate comparison for the docking of tRNAPhe in the two models, the tyrosyl-tRNA synthetase dimers from each model have been overlaid. As a result, only a single tyrosyl-tRNA synthetase dimer is seen in (a) and (b). In (a), the tyrosyl-tRNA synthetase:tRNAPhe complexes are shown in side view, while in (b), the complexes have been rotated by 90  about the x-axis and 45  about the y-axis to present a top view of the two models. In both (a) and (b), chains A and B of the tyrosyl-tRNA synthetase dimer are shown as orange and cyan ribbons, respectively, and the tyrosyl-adenylate intermediate is shown as a green-colored stick representation. tRNAPhe is shown as a purple ribbon representation for Bedouelle's model and as a black ribbon representation for the model proposed here. The KMSKS motif in the anticodon loop-recognition subunit of tyrosyl-tRNA synthetase is shown as a red stick representation. In (c), a close-up of the interaction between adenosine 76 in tRNA Phe and Thr40, Lys82, and Arg86 in tyrosyl-tRNA synthetase is shown. Adenosine 76, Thr40, Lys82, and Arg86 are shown as stick representations with the following color scheme: oxygen, red; carbon, green; nitrogen, blue; hydrogen, gray. Putative hydrogen bonds are shown as black lines.

sine 76 is sandwiched between Thr40 on one side and Lys82 and Arg86 on the other side. Both the results and modeling studies presented here support the model proposed by Bedouelle et al. for the binding of tRNATyr in the active site of tyrosyl-tRNA synthetase (Bedouelle & Winter, 1986; Bedouelle, 1990; Bedouelle et al., 1993). Speci®cally, the data presented here are consistent with a model in which Thr40 and Lys82 both form single hydrogen bonds with adenosine 76, while Arg86 forms two hydrogen bonds with adenosine 76. Based on the modeling studies, it appears likely

that formation of these hydrogen bonds involves minor conformational changes within the active site of the enzyme during formation of the transition state. This is consistent with Thr40, Lys82, and Arg86 each having a small effect on the initial binding of tRNATyr (0.5-1.0 kcal/mol) and a much larger effect (1.7-3.5 kcal/mol) on the stability of the transition state. The primary role of these amino acids appears to be ensuring that the A76 nucleotide in tRNATyr is properly oriented for attack on the carbonyl group of the tyrosyl-adenylate intermediate.

Evolution of Catalysis in Tyrosyl-tRNA Synthetase

The roles played by Thr40, Lys82, and Arg86 in the second step of tRNA aminoacylation mimic their roles in the first step of the aminoacylation reaction Extensive mutagenesis and kinetic analyses on the ®rst step of the tRNATyr aminoacylation reaction have shown that while the interactions between tyrosyl-tRNA synthetase and the tyrosine substrate remain relatively constant throughout the course of the ®rst step of the reaction, the interactions between tyrosyl-tRNA synthetase and the ATP substrate increase in strength as the reaction proceeds, reaching a maximum in the E
[TyrATP]{ complex (reviewed by Fersht, 1987). This earlier work provides strong experimental support for the hypothesis that enzymes use binding energy to preferentially stabilize the transition state of a reaction (Haldane, 1930; Pauling, 1946). In particular, Fersht and colleagues observed that Thr40, Lys82, and Arg86 contribute little binding energy during formation of the initial E
Tyr
ATP complex (Leatherbarrow et al., 1985; Leatherbarrow & Fersht, 1987; Fersht et al., 1988). On formation of the transition state complex, however, these groups each contribute 3.0-5.3 kcal/mol of binding energy towards the stability of the E
[Tyr-ATP]{ complex. A similar situation exists in the second step of the aminoacylation reaction. Thr40, Lys82, and Arg86 each contribute only modest amounts of binding energy (<1 kcal/mol) in the E
Tyr-AMP
tRNATyr complex preceding the transition state. During formation of the E
[Tyr-tRNATyr
AMP]{ complex, however, these amino acid residues each contribute 1.7-3.4 kcal/mol of binding energy. In other words, in the second step of the reaction, the interaction between adenosine 76 and the Thr40, Lys82, and Arg86 side-chains reaches its maximum on formation of the transition state complex. A model for the evolution of the aminoacyl-tRNA synthetases It has been postulated that the optimization of catalysis in tyrosyl-tRNA synthetase has been achieved by balancing the activation energy barriers for each step in the aminoacylation reaction (Avis & Fersht, 1993). In this model, when two or more transition states exist, the rate of the reaction is signi®cantly increased only when the higherenergy transition state is stabilized (Albery & Knowles, 1976). Once the transition state for the initial rate-limiting step is stabilized to the point where it is no longer rate-limiting, increasing its stability further will have little or no effect on the rate of the reaction. At this point, a further increase in catalytic activity requires stabilization of the next-highest transition state. This process continues with ®rst one transition state being stabilized, then the other, until the catalytic activity has been optimized for the overall reaction. As a result, when two or more transition states are involved, optimization of catalytic activity produces transition

293 states with nearly identical activation energy barriers (Albery & Knowles, 1976). This is the situation that exists in tyrosyl-tRNA synthetase, with the activation barrier for each of the steps in the reaction being 15.3 kcal/mol (Avis & Fersht, 1993). By correlating the conservation of each activesite residue with its role in catalysis, we can get a picture of how the catalytic mechanism of tyrosyltRNA synthetase has evolved. The active-site amino acid residues in B. stearothermophilus tyrosyl-tRNA synthetase can be grouped into one of ®ve categories, based on their conservation in other organisms (Figure 5): (1) amino acids that are highly conserved among all of the class I aminoacyl-tRNA synthetases; (2) amino acids that are highly conserved in a subset of the class I amino-

Figure 5. Phylogenetic tree showing the evolution of active site residues in B. stearothermophilus tyrosyl-tRNA synthetase. Classi®cation of amino acids into those conserved in class I aminoacyl-tRNA synthetases and those conserved in subsets of the class I aminoacyl-tRNA synthetases is based on the sequence alignment presented by Landes et al. (1995). Classi®cation of amino acids into those conserved in all tyrosyl-tRNA synthetase, bacterial tyrosyl-tRNA synthetases, and B. stearothermophilus tyrosyl-tRNA synthetase is based on alignment of 24 bacterial tyrosyl-tRNA synthetase sequences (Aquifex aeolicus, Bacillus caldotenax, Bacillus stearothermophilus, Bacillus subtilis isozymes 1 and 2, Borrelia burgdorferi, Chlamydia pneumoniae, Chlamydia trachomatis, Deinococcus radiodurans, Escherichia coli, Haemophilus in¯uenzae, Helicobacter pylori, Helicobacter pylori j99, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma genitalium, Mycoplasma pneumoniae, Rickettsia prowazekii, Streptomyces coelicor, Synechocystis species pcc5803, Thermotoga maritama, Thiobacillus ferrooxidans, Treponema pallidum, Ureaplasma urealyticum), eight eukaryotic tyrosyl-tRNA synthetase sequences (Bos taurus, Caenorhabditis elegans, Candida albicans, Drosophila melangaster, Drosophila virilis, Homo sapiens, Saccharomyces cerevisiae, Schizosaccharomyces pombe), and seven archaeal tyrosyl-tRNA synthetase sequences (Aeropyrum pernix, Archeaoglobus fulgidus, Methanococcus thermautotropicum, Methanococcus jannaschii, Pyrococcus abysii, Pyrococcus horikoshii, Sulfolobus solfataricus) (E.A.F., unpublished results).

294 acyl-tRNA synthetases (e.g. tyrosyl, tryptophanyl, glutaminyl, and glutamyl-tRNA synthetases); (3) amino acids that are highly conserved among all of the tyrosyl-tRNA synthetases; (4) amino acids that are highly conserved among only bacterial tyrosyltRNA synthetases; and (5) amino acids that are present only in a small subset of the bacterial tyrosyl-tRNA synthetases. Presumably, category (1) corresponds to the amino acids that appeared early in the evolution of the class I aminoacyl-tRNA synthetases, whereas category (5) corresponds to the amino acids that arose relatively late in the evolutionary history of the enzyme. Examining the functions of the amino acids in each group suggests the following evolutionary history for the active site of tyrosyl-tRNA synthetase from B. stearothermophilus: the ®rst catalytic residues to appear were those in the class I conserved HIGH and KMSKS sequence motifs. Based on the role that these motifs play in the catalytic mechanism of tyrosyl-tRNA synthetase, we propose that stabilizing the transition state for formation of the aminoacyl-adenylate species was the initial function of the primordial aminoacyl-tRNA synthetases. This is discussed in more detail below. Category (2) includes the amino acids that recognize the protonated ammonium group of the tyrosine substrate (e.g. Asp78 and Tyr169; Wells & Fersht, 1986; Lowe et al., 1987; Fersht et al., 1988; de Prat Gay et al., 1993), suggesting that amino acid binding arose after the evolution of catalytic activity with respect to aminoacyl-adenylate formation. The observation that Asp78 and Tyr169 interact with the protonated ammonium group of the tyrosine substrate, rather than its side-chain, is consistent with the hypothesis that the amino acid speci®city of the primordial aminoacyl-tRNA synthetases was fairly broad (Stathopoulos et al., 2000). The third and fourth categories contain amino acids involved in stabilization of the transition state for the second step of the reaction (e.g. Lys82, Arg86, and Gln173; results presented here and in the accompanying paper), and anticodon recognition (e.g. Lys410 and Lys411; Bedouelle & Winter, 1986). This suggests that the development of amino acid binding preceded anticodon recognition and catalysis of the second step of the reaction. Finally, the ®fth category includes amino acid residues such as Cys35 and Thr51, whose presumed role is to optimize the catalytic ef®ciency of tyrosyl-tRNA synthetase for growth conditions that are speci®c to B. stearothermophilus (Wilkinson et al., 1983; Fersht et al., 1985; Lowe et al., 1985; Ho & Fersht, 1986; Jones et al., 1986; Wells & Fersht, 1986). Based on the results presented here, as well as previous analyses of the roles of the HIGH and KMSKS motifs in catalysis (Leatherbarrow et al., 1985; Lowe et al., 1985; Jones et al., 1986; Leatherbarrow & Fersht, 1987; Fersht et al., 1988; First & Fersht, 1993a,b,c, 1995; Xin et al., 2000a), we propose that the initial step in the evolution of catalytic activity in tyrosyl-tRNA synthetase was stabilization of the transition state for the acti-

Evolution of Catalysis in Tyrosyl-tRNA Synthetase

vation of the tyrosine substrate. This hypothesis is based on the following lines of reasoning. Both the HIGH and KMSKS motifs are highly conserved among the class I aminoacyl-tRNA synthetases. This is consistent with the appearance of the HIGH and KMSKS motifs being an early event during the evolution of these enzymes. Data presented here and by Xin et al. (2000a) indicate that neither the HIGH motif nor the KMSKS motif stabilizes the transition state for the second step of the tRNA aminoacylation reaction. In other words, the HIGH and KMSKS motifs stabilize only the transition state for the activation of tyrosine, suggesting that catalysis of the amino acid activation step was an early event in the evolution of the aminoacyl-tRNA synthetases. The hypothesis that this event occurred prior to stabilization of the transition state for the second step of the aminoacylation reaction is supported by the observation that Thr40, Lys82, Arg86, and Gln173 all arose after the class I aminoacyl-tRNA synthetases had begun to diverge (Figure 5). It is intriguing that Thr40, Lys82, and Arg86 stabilize both steps of the aminoacylation reaction. This suggests that their initial role may have been to stabilize the transition state for the formation of tyrosyl-adenylate. Once the transition state for the second step of the reaction became rate-limiting, these three amino acid residues were presumably co-opted to help catalyze the transfer of tyrosine to tRNATyr. Based on sequence analysis of the class II seryltRNA synthetase, Hartlein & Cusack (1995) postulated that the aminoacyl-tRNA synthetases are descended from enzymes whose function was to activate small molecules with ATP. This hypothesis is consistent with the results presented here, which indicate that the most highly conserved amino acids in the class I aminoacyl-tRNA synthetases interact with the pyrophosphate moiety of ATP. If the above hypothesis is correct, it raises the possibility that the primordial aminoacyl-tRNA synthetases did not catalyze the aminoacylation of RNA substrates, but instead synthesized and released the aminoacyl-adenylate species as the ®nal product of the reaction. This hypothesis is consistent with the proposal that recognition of the tRNA anticodon was a relatively late event in the evolution of the aminoacyl-tRNA synthetases (Francklyn & Schimmel, 1989; Schwob & SoÈll, 1993; Lipman & Hou, 1998; Siatecka et al., 1998; Wakasugi et al., 1998; Steer & Schimmel, 1999). It is also consistent with the results of Fersht and colleagues, who observed that substitution of Asp78 with alanine in tyrosyl-tRNA synthetase results in the premature release of the tyrosyl-adenylate intermediate from the enzyme (Lowe et al., 1987), suggesting that tyrosyl-adenylate was not tightly bound in the primordial tyrosyl-tRNA synthetase. If the original function of the aminoacyl-tRNA synthetases was solely to activate amino acids, what were these activated amino acids used for? The ``RNA world'' hypothesis postulates that template-dependent protein translation was initially

295

Evolution of Catalysis in Tyrosyl-tRNA Synthetase

catalyzed by ribozymes (Gilbert, 1986). The data presented here are consistent with a scenario in which the primordial aminoacyl-tRNA synthetases initially replaced a ribozyme that catalyzed the activation of amino acids used in protein synthesis, then subsequently replaced a ribozyme that catalyzed the transfer of the activated amino acid to the 30 end of tRNA. It has been proposed that in the RNA world, template-independent peptide synthesis preceded the development of templatedependent peptide synthesis (Lipmann, 1965, 1971; Reanney, 1977; de Duve 1987; Danchin, 1989; Orgel, 1989; Wong, 1991; Schimmel & Henderson, 1994; DiGiulio, 1994, 1996). If this hypothesis is correct, it is possible that the HIGH and KMSKS motifs are descendants of peptides that were initially synthesized by a template-independent mechanism. The central role of these two motifs in catalyzing the ®rst step of the tRNA aminoacylation reaction suggests that their ancestral peptides may have been coenzymes used by a ribozyme that catalyzed the activation of amino acids and other small molecules. If this hypothesis is correct, it suggests that these ancestral peptides played a central role in both protein synthesis and the metabolism of small molecules in the RNA world.

Materials and Methods Materials Reagents and vectors were purchased from the following sources: Taq and Pfu DNA polymerases, T4 DNA Ligase, Wizard PCR prep, Plasmid prep, and DNA Cleanup systems from Promega; NuSieve low melting point agarose from FMC BioProducts; L-[14C]tyrosine from Moravek Biochemicals; NAP 25 columns from Pharmacia; XL1 Blue competent cells from Stratagene; and oligonucleotides from Life Technologies Inc. Automated DNA sequencing was performed by the DNA sequencing facility at Iowa State University using dyelabeled dideoxy terminators. All other chemicals and reagents were purchased from Fisher Scienti®c. Site-directed mutagenesis of tyrosyl-tRNA synthetase All tyrosyl-tRNA synthetase variants were constructed by a two-step PCR mutagenesis procedure (Higuchi et al., 1988). The pYTS5-WT phagemid, a derivative of pTZ18u that contains the wild-type tyrosyl-tRNA synthetase gene from B. stearothermophilus, was used as the template for the initial PCR mutagenesis reaction (First & Fersht, 1993a). The following nucleotides were used to create the desired mutations (mismatches are in italics): 50 C GGG TTT GAC CCA GCT GCG GAC AGT TTG 30 (T40A with a PvuII restriction site), 50 CG GCG GAC AGT CTC GCG ATC GGC CADC TTG GCC A 30 (H45A with a NruI restriction site), 50 GT TTG CAT ATC GGC GCG CTA GCC ACC ATT TTG 30 (H48A with a NheI restriction site), 50 GAC CCG AGC GGC GCC AAA AGC GAG CGC 30 (K82A with a NarI restriction site), 50 C GGG AAA AAA AGC GAG GCC ACT TTA AAT GCC AAA GAA ACC G 30 (R86A with a DraI restriction site). None of the restriction sites introduced altered the amino acid sequence of the ®nal protein product. Two

additional primers, 50 CTG TCG GGT TTA GCC ACC TCT GAC 30 and 50 AGG GCG ATG GCC CAC TAC GTG 30 , were used as the outside primers in the PCR mutagenesis reactions. These primers are complementary to the pTZ18u vector sequence. The templates and PCR products were puri®ed with Promega Wizard PCR Preps DNA Puri®cation and CleanUp Systems. PCR products containing the variant tyrosyl-tRNA synthetase coding sequences were digested with BamHI and HindIII restriction enzymes and subcloned into a BamHI/HindIII fragment of the pYTS5-WT plasmid that lacks the tyrosyltRNA synthetase coding sequence (First & Fersht, 1993a). Positive clones were selected by PCR ampli®cation of the tyrosyl-tRNA synthetase coding sequence followed by digestion with the restriction endonuclease corresponding to the site introduced during the PCR mutagenesis reaction. The entire tyrosyl-tRNA synthetase coding sequence for each variant was veri®ed by automated DNA sequencing. Purification of tyrosyl-tRNA synthetase and the tRNATyr substrate Puri®cation of the wild-type and variant tyrosyl-tRNA synthetases has been described (First & Fersht, 1993a; Xin et al., 2000a). tRNATyr substrate was obtained by in vitro transcription from a FokI-linearized pGFX-WT plasmid as described (Avis et al., 1993; Xin et al., 2000a). In vitro analysis of the tyrosyl-tRNA synthetase variants: pre-steady state kinetic measurements of tRNATyr aminoacylation Formation of the E
[Tyr-tRNATyr
AMP]{ complex is accompanied by a increase in the intrinsic ¯uorescence of the protein (Avis et al., 1993). An Applied Photophysics model SX 18.MV stopped-¯ow spectrophotometer was used to monitor changes in the intrinsic ¯uorescence of the E
Tyr-AMP intermediate on the addition of tRNATyr as described (Avis et al., 1993; Xin et al., 2000a). Brie¯y, the procedure is as follows: The E
Tyr-AMP intermediate is preformed by incubating tyrosyl-tRNA synthetase with 10 mM MgATP and 100 mM tyrosine at 25  C and pH 7.78 for 30-45 minutes. The E
Tyr-AMP intermediate is then isolated by chromatography on a Nap 25 Column (Pharmacia). This complex is mixed with various concentrations of in vitro transcribed tRNATyr substrate in the stopped-¯ow ¯uorometer and the change in the intrinsic ¯uorescence of the protein is monitored over time using an excitation wavelength of 295 nm and an emission ®lter with a cutoff above 320 nm. A rapid equilibrium assumption is applied for the tyrosine transfer step of the aminoacylation reaction (Avis et al., 1993). As a consequence of this assumption, formation of the transition state complex is the rate-limiting step for the second step of the aminoacylation reaction. All of the experiments were done at least three times with both tyrosyl-tRNA synthetase and tRNATyr substrate from at least two different protein preparations or in vitro transcription reactions. A least-squares analysis is used to ®t the rate curves to a single exponential equation with ¯oating end point and the initial rate was calculated by extrapolation to time zero. For each variant, the rate and dissociation constants (k4 and KtRNA) are obtained by ®tting the substrate dependence of the initial rate to the Michaelis-Menten equation using a non-linear regression method (Leatherbarrow, 1990).

296

Evolution of Catalysis in Tyrosyl-tRNA Synthetase

Calculation of free energies of binding

References

The following equations were used to calculate the relative free energies for the formation of the E
TyrAMP
tRNATyr and E
[Tyr-tRNATyr
AMP]{ complexes (Avis et al., 1993):

Albery, W. J. & Knowles, J. R. (1976). Evolution of enzyme function and the development of catalytic ef®ciency. Biochemistry, 15, 5631-5640. Avis, J. M. & Fersht, A. R. (1993). Use of binding energy in catalysis: optimization of rate in a multistep reaction. Biochemistry, 32, 5321-5326. Avis, J. M., Day, A. G., Garcia, G. A. & Fersht, A. R. (1993). Reaction of modi®ed and unmodi®ed tRNATyr substrates with tyrosyl-tRNA synthetase (Bacillus stearothermophilus). Biochemistry, 32, 53125320. Bedouelle, H. (1990). Recognition of tRNATyr by tyrosyltRNA synthetase. Biochimie, 72, 589-598. Bedouelle, H. & Winter, G. (1986). A model of synthetase/transfer RNA interaction as deduced by protein engineering. Nature, 320, 371-373. Bedouelle, H., Guez-Ivanier, V. & Nageotte, R. (1993). Discrimination between transfer-RNAs by tyrosyltRNA synthetase. Biochimie, 75, 1099-1108. Borgford, T. J., Brand, N. J., Gray, T. E. & Fersht, A. R. (1987). The valyl-tRNA synthetase from Bacillus stearothermophilus has considerable sequence homology with the isoleucyl-tRNA synthetase from Escherichia coli. Biochemistry, 26, 2480-2486. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Brick, P. & Blow, D. M. (1987). Crystal structure of a deletion mutant of a tyrosyl-tRNA synthetase complexed with tyrosine. J. Mol. Biol. 194, 287-297. Brick, P., Bhat, T. N. & Blow, D. M. (1989). Structure of Ê resolution. tyrosyl-tRNA synthetase re®ned at 2.3 A Interaction of the enzyme with the tyrosyl-adenylate intermediate. J. Mol. Biol. 208, 83-98. Carter, P., Bedouelle, H. & Winter, G. (1986). Construction of heterodimer tyrosyl-tRNA synthetase shows tRNATyr interacts with both subunits. Proc. Natl Acad. Sci. USA, 83, 1189-1192. Cusack, S., Berthet-Colominas, C., Hartlein, M., Nassar, N. & Leberman, R. (1990). A second class of synthetase structure revealed by X-ray analysis of EscheriÊ . Nature, 347, chia coli seryl-tRNA synthetase at 2.5 A 249-255. Danchin, A. (1989). Homeotopic transformation and the origin of translation. Prog. Biophys. Mol. Biol. 54, 8186. de Duve, C. (1987). Selection by differential molecular survival: a possible mechanism of early chemical evolution. Proc. Natl Acad. Sci. USA, 84, 8253-8256. de Prat Gay, G., Duckworth, H. W. & Fersht, A. R. (1993). Modi®cation of the amino acid speci®city of tyrosyl-tRNA synthetase by protein engineering. FEBS Letters, 318, 167-171. Di Giulio, M. (1994). On the origin of protein synthesis: a speculative model based on hairpin RNA structures. J. Theor. Biol. 171, 303-308. Di Giulio, M. (1996). The origin of protein synthesis: on some molecular fossils identi®ed through comparison of protein sequences. Biosystems, 39, 159-169. Dwyer, D. S. (1996). Molecular model of interleukin 12 that highlights amino acid sequence homologies with adhesion domains and gastrointestinal peptides. J. Mol. Graph. 14, 148-157. Dwyer, D. S. (1999). Molecular simulation of the effects of alcohol on peptide structure. Biopolymers, 49, 635645.


GE
Tyr-AMP
tRNA ˆ RT ln…KtRNA †

…3†


GE
‰Tyr-tRNA
AMPŠz ˆ RT ln…kB T=h† ÿ RT ln…k4 =KtRNA † …4† where E
Tyr-AMP
tRNATyr is an intermediate complex preceding the formation of the transition state for the second step of the tRNATyr aminoacylation reaction, E
[Tyr-tRNATyr
AMP]{ is the transition state complex for the second step, kB is Boltzmann's constant, T is the temperature in K, h is Planck's constant and R is the gas constant. Free energy changes calculated using equations (3) and (4) are relative to the free energy change for E
Tyr-AMP complex. Molecular modeling between tyrosyl-tRNA synthetase and yeast tRNAPhe The interaction between tyrosyl-tRNA synthetase and tRNAPhe was modeled using a Silicon Graphics Indigo2 Extreme workstation with Insight II software from Molecular Simulations, Inc. (San Diego, CA) as described (Dwyer, 1996, 1999). The model was created using tRNAPhe (Protein Data Bank accession ®le, 1tra; Westhof & Sundaralingam, 1986) and tyrosyl-tRNA synthetase (accession number 4ts1; Brick & Blow, 1987). In order to localize tyrosyl-adenylate in the model, the structure of monomeric tyrosyl-tRNA synthetase (3ts1; Brick et al., 1989), which includes the bound tyrosinyl-adenylate inhibitor, was superimposed on the dimer form, tyrosyltRNA synthetase (4ts1). As a ®rst approximation, the tRNAPhe molecule was positioned so that the stem region ®t into the acceptor-binding site of the dimer, whereas the anticodon arm approached the KMSKS loop in the second subunit of the protein. The interface between the two molecules was then de®ned as the subset of amino Ê of Arg86, Glu152, Ser156, Gly192, acids within 4 A Ser193, Asp194, Gln195, Trp196, and Leu222. Binding energies (van der Waals forces) between the probe (stem region 71-76) and the interface subset were monitored using the Docking module of the Insight software. On the basis of this information, tRNAPhe was maneuvered into position for optimum binding. Nucleotide C75 was then manually rotated to avoid steric clashes with the side-chains of the protein and A76 was rotated to position it in close proximity to the tyrosinyl-adenylate inhibitor in the co-crystal structure. The model of the binding interaction was further re®ned after each of these steps using the docking energy as a guide.

Acknowledgments We thank Professor Alan Fersht for the pGAG2 plasmid and Dr George Garcia for the Escherichia coli TG2 strain, Dr Charles Carter for helpful discussions concerning various aspects of this work, and Dr Hugues Bedouelle for providing the coordinates for the TyrRS
tRNAPhe model and for communicating unpublished results. This work is supported by National Institutes of Health grant GM53693.

Evolution of Catalysis in Tyrosyl-tRNA Synthetase Eiler, S., Dock-Bregeon, A., Moulinier, L., Thierry, J. C. & Moras, D. (1999). Synthesis of aspartyltRNA(Asp) in Escherichia coli ± a snapshot of the second step. EMBO J. 18, 6532-6541. Eriani, G., Dirheimer, G. & Gangloff, J. (1990). Structurefunction relationship of arginyl-tRNA synthetase from Escherichia coli: isolation and characterization of the argS mutation MA5002. Nucl. Acids Res. 18, 1475-1479. Fersht, A. R. (1987). Dissection of the structure and activity of the tyrosyl-tRNA synthetase by sitedirected mutagenesis. Biochemistry, 26, 8031-8037. Fersht, A. R., Wilkinson, A. J., Carter, P. & Winter, G. (1985). Fine structure-activity analysis of mutations at position 51 of tyrosyl-tRNA synthetase. Biochemistry, 24, 5858-5861. Fersht, A. R., Knill-Jones, J. W., Bedouelle, H. & Winter, G. (1988). Reconstruction by site-directed mutagenesis of the transition state for the activation of tyrosine by the tyrosyl-tRNA synthetase: a mobile loop envelopes the transition state in an induced-®t mechanism. Biochemistry, 27, 1581-1587. First, E. A. & Fersht, A. R. (1993a). Involvement of threonine 234 in catalysis of tyrosyl adenylate formation by tyrosyl-tRNA synthetase. Biochemistry, 32, 13644-13650. First, E. A. & Fersht, A. R. (1993b). Mutation of lysine 233 to alanine introduces positive cooperativity into tyrosyl-tRNA synthetase. Biochemistry, 32, 1365113657. First, E. A. & Fersht, A. R. (1993c). Mutational and kinetic analysis of a mobile loop in tyrosyl-tRNA synthetase. Biochemistry, 32, 13658-13663. First, E. A. & Fersht, A. R. (1995). Analysis of the role of the ``KMSKS'' loop in the catalytic mechanism of the tyrosyl-tRNA synthetase using multimutant cycles. Biochemistry, 34, 5030-5043. Francklyn, C. & Schimmel, P. (1989). Aminoacylation of RNA minihelices with alanine. Nature, 337, 478-481. Gilbert, W. (1986). The RNA world. Nature, 319, 618. Goldgur, Y., Mosyak, L., Reshetnikova, L., Ankilova, V., Lavrik, O., Khodyreva, S. & Safro, M. (1997). The crystal structure of phenylalanyl-tRNA synthetase from Thermus thermophilus complexed with cognate tRNAPhe. Structure, 5, 59-68. Haldane, J. B. S. (1930). Enzymes, Longmans, Green and Co., London M.I.T. Press, Boston (1965). Hartlein, M. & Cusack, S. (1995). Structure, function and evolution of seryl-tRNA synthetases: implications for the evolution of aminoacyl-tRNA synthetases and the genetic code. J. Mol. Evol. 40, 519-530. Higuchi, R., Krummel, B. & Saiki, R. K. (1988). A general method of in vitro preparation and speci®c mutagenesis of DNA fragments: study of protein and DNA interactions. Nucl. Acids Res. 16, 73517367. Hingerty, B., Brown, R. S. & Jack, A. (1978). Further re®nement of the structure of yeast tRNAPhe. J. Mol. Biol. 124, 523-534. Ho, C. K. & Fersht, A. R. (1986). Internal thermodynamics of position 51 mutants and natural variants of tyrosyl-tRNA synthetase. Biochemistry, 25, 18911897. Hountondji, C., Dessen, P. & Blanquet, S. (1986). Sequence similarities among the family of aminoacyl-tRNA synthetases. Biochimie, 68, 1071-1078. Jack, A., Ladner, J. E. & Klug, A. (1976). Crystallographic re®nement of yeast phenylalanine transfer Ê resolution. J. Mol. Biol. 108, 619-649. RNA at 2.5 A

297 Jakes, R. & Fersht, A. R. (1975). Tyrosyl-tRNA synthetase from Escherichia coli. Stoichiometry of ligand binding and half-of-the-sites reactivity in aminoacylation. Biochemistry, 14, 3344-3350. Jones, M. D., Lowe, D. M., Borgford, T. & Fersht, A. R. (1986). Natural variation of tyrosyl-tRNA synthetase and comparison with engineered mutants. Biochemistry, 25, 1887-1891. Landes, C., Perona, J. J., Brunie, S., Rould, M. A., Zelwer, C., Steitz, T. A. & Risler, J. L. (1995). A structure-based multiple sequence alignment of all class I aminoacyl-tRNA synthetases. Biochimie, 77, 194-203. Leatherbarrow, R. J. (1990). Using linear and non-linear regression to ®t biochemical data. Trends Biochem. Sci. 15, 455-458. Leatherbarrow, R. J. & Fersht, A. R. (1987). Investigation of transition-state stabilization by residues histidine45 and threonine-40 in the tyrosyl-tRNA synthetase. Biochemistry, 26, 8524-8528. Leatherbarrow, R. J., Fersht, A. R. & Winter, G. (1985). Transition-state stabilization in the mechanism of tyrosyl-tRNA synthetase revealed by protein engineering. Proc. Natl Acad. Sci. USA, 82, 7840-7844. Lipman, R. S. & Hou, Y. M. (1998). Aminoacylation of tRNA in the evolution of an aminoacyl-tRNA synthetase. Proc. Natl Acad. Sci. USA, 95, 1349513500. Lipmann, F. (1965). Projecting backward from the present stage of evolution of biosynthesis. In Origins of Prebiological Systems and of their Molecular Matrices. (Fox, S., ed.), pp. 259-280, Academic Press, New York. Lipmann, F. (1971). Attempts to map a process evolution of peptide biosynthesis. Science, 173, 875-884. Lowe, D. M., Fersht, A. R., Wilkinson, A. J., Carter, P. & Winter, G. (1985). Probing histidine-substrate interactions in tyrosyl-tRNA synthetase using asparagine and glutamine replacements. Biochemistry, 24, 51065109. Lowe, D. M., Winter, G. & Fersht, A. R. (1987). Structure-activity relationships in engineered proteins: characterization of disruptive deletions in the alphaammonium group binding site of tyrosyl-tRNA synthetase. Biochemistry, 26, 6038-6043. Orgel, L. E. (1989). The origin of polynucleotide-directed protein synthesis. J. Mol. Evol. 29, 465-474. Pauling, L. (1946). Molecular architecture and biological reactions. Chem. Eng. News, 24, 1375-1377. Perona, J. J., Rould, M. A. & Steitz, T. A. (1993). Structural basis for transfer RNA aminoacylation by Escherichia coli glutaminyl-tRNA synthetase. Biochemistry, 32, 8758-8771. Reanney, D. C. (1977). Aminoacyl thiol esters and the origins of genetic speci®city. J. Theor. Biol. 65, 555569. Rossmann, M. G., Moras, D. & Olsen, K. W. (1974). Chemical and biological evolution of nucleotidebinding protein. Nature, 250, 194-199. Sankaranarayanan, R., Dock-Bregeon, A. C., Romby, P., Caillet, J., Springer, M., Rees, B., Ehresmann, C., Ehresmann, B. & Moras, D. (1999). The structure of threonyl-tRNA synthetase-tRNA(Thr) complex enlightens its repressor activity and reveals an essential zinc ion in the active site. Cell, 97, 371-381. Schimmel, P. & Henderson, B. (1994). Possible role of aminoacyl-RNA complexes in noncoded peptide synthesis and origin of coded synthesis. Proc. Natl Acad. Sci. USA, 91, 11283-11286.

298

Evolution of Catalysis in Tyrosyl-tRNA Synthetase

Schwob, E. & SoÈll, D. (1993). Selection of a ``minimal'' glutaminyl-tRNA synthetase and the evolution of class I synthetases. EMBO J. 12, 5201-5208. Siatecka, M., Rozek, M., Barciszewski, J. & Mirande, M. (1998). Modular evolution of the Glx-tRNA synthetase family ± rooting of the evolutionary tree between the bacteria and archaea/eukarya branches. Eur. J. Biochem. 256, 80-87. Silvian, L. F., Wang, J. & Steitz, T. A. (1999). Insights into editing from an Ile-tRNA synthetase structure with tRNAIle and mupirocin. Science, 285, 10741077. Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H. D., Ibba, M. & SoÈll, D. (2000). One polypeptide with two aminoacyl-tRNA synthetase activities. Science, 287, 479-482. Steer, B. A. & Schimmel, P. (1999). Major anticodonbinding region missing from an archaebacterial tRNA synthetase. J. Biol. Chem. 274, 35601-35606. Wakasugi, K., Quinn, C. L., Tao, N. & Schimmel, P. (1998). Genetic code in evolution: switching speciesspeci®c aminoacylation with a peptide transplant. EMBO J. 17, 297-305. Waye, M. M., Winter, G., Wilkinson, A. J. & Fersht, A. R. (1983). Deletion mutagenesis using an `M13 splint': the N-terminal structural domain of tyrosyl-tRNA synthetase (B. stearothermophilus) catalyses the formation of tyrosyl-adenylate. EMBO J. 2, 1827-1829.

Webster, T., Tsai, H., Kula, M., Mackie, G. A. & Schimmel, P. (1984). Speci®c sequence homology and three-dimensional structure of an aminoacyltransfer RNA synthetase. Science, 226, 1315-1317. Wells, T. N. & Fersht, A. R. (1986). Use of binding energy in catalysis analyzed by mutagenesis of the tyrosyl-tRNA synthetase. Biochemistry, 25, 18811886. Westhof, E. & Sundaralingam, M. (1986). Restrained re®nement of the monoclinic form of yeast phenylalanine transfer RNA. Temperature factors and dynamics, coordinated waters, and base-pair propeller twist angles. Biochemistry, 25, 4868-4878. Wilkinson, A. J., Fersht, A. R., Blow, D. M. & Winter, G. (1983). Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation. Biochemistry, 22, 3581-3586. Wong, J. T. (1991). Origin of genetically encoded protein synthesis: a model based on selection for RNA peptidation. Orig. Life Evol. Biosph. 21, 165-176. Xin, Y., Li, W. & First, E. A. (2000a). The ``KMSKS'' motif in tyrosyl-tRNA synthetase participates in the initial binding of tRNATyr. Biochemistry, 39, 340-347. Xin, Y., Li, W. & First, E. A. (2000b). Stabilization of the transition state for the transfer of tyrosine to tRNATyr by tyrosyl-tRNA synthetase. J. Mol. Biol. 303, 299-310.

Edited by A. R. Fersht (Received 12 April 2000; received in revised form 22 August 2000; accepted 22 August 2000)