Stabilization of the transition state for the transfer of tyrosine to tRNATyr by tyrosyl-tRNA synthetase1 (original) (raw)
Related papers
J Mol Biol, 2000
Aminoacylation of tRNA Tyr involves two steps: (1) tyrosine activation to form the tyrosyl-adenylate intermediate; and (2) transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA Tyr. In Bacillus stearothermophilus tyrosyl-tRNA synthetase, Asp78, Tyr169, and Gln173 have been shown to form hydrogen bonds with the a-ammonium group of the tyrosine substrate during the ®rst step of the aminoacylation reaction. Asp194 and Gln195 stabilize the transition state complex for the ®rst step of the reaction by hydrogen bonding with the 2 H-hydroxyl group of AMP and the carboxylate oxygen atom of tyrosine, respectively. Here, the roles that Asp78, Tyr169, Gln173, Asp194, and Gln195 play in catalysis of the second step of the reaction are investigated. Pre-steady-state kinetic analyses of alanine variants at each of these positions shows that while the replacement of Gln173 by alanine does not affect the initial binding of the tRNA Tyr substrate, it destabilizes the transition state complex for the second step of the reaction by 2.3 kcal/mol. None of the other alanine substitutions affects either the initial binding of the tRNA Tyr substrate or the stability of the transition state for the second step of the aminoacylation reaction. Taken together, the results presented here and the accompanying paper are consistent with a concerted reaction mechanism for the transfer of tyrosine to tRNA Tyr , and suggest that catalysis of the second step of tRNA Tyr aminoacylation involves stabilization of a transition state in which the scissile acylphosphate bond of the tyrosyl-adenylate species is strained. Cleavage of the scissile bond on the breakdown of the transition state alleviates this strain.
Journal of Molecular Biology, 2000
Aminoacylation of tRNA Tyr involves two steps: (1) tyrosine activation to form the tyrosyl-adenylate intermediate; and (2) transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA Tyr. In Bacillus stearothermophilus tyrosyl-tRNA synthetase, Asp78, Tyr169, and Gln173 have been shown to form hydrogen bonds with the a-ammonium group of the tyrosine substrate during the ®rst step of the aminoacylation reaction. Asp194 and Gln195 stabilize the transition state complex for the ®rst step of the reaction by hydrogen bonding with the 2 H-hydroxyl group of AMP and the carboxylate oxygen atom of tyrosine, respectively. Here, the roles that Asp78, Tyr169, Gln173, Asp194, and Gln195 play in catalysis of the second step of the reaction are investigated. Pre-steady-state kinetic analyses of alanine variants at each of these positions shows that while the replacement of Gln173 by alanine does not affect the initial binding of the tRNA Tyr substrate, it destabilizes the transition state complex for the second step of the reaction by 2.3 kcal/mol. None of the other alanine substitutions affects either the initial binding of the tRNA Tyr substrate or the stability of the transition state for the second step of the aminoacylation reaction. Taken together, the results presented here and the accompanying paper are consistent with a concerted reaction mechanism for the transfer of tyrosine to tRNA Tyr , and suggest that catalysis of the second step of tRNA Tyr aminoacylation involves stabilization of a transition state in which the scissile acylphosphate bond of the tyrosyl-adenylate species is strained. Cleavage of the scissile bond on the breakdown of the transition state alleviates this strain.
Journal of Biological Chemistry, 2008
Tyrosyl-tRNA synthetase (TyrRS) is able to catalyze the transfer of both Land D-tyrosine to the 3 end of tRNA Tyr. Activation of either stereoisomer by ATP results in formation of an enzyme-bound tyrosyl-adenylate intermediate and is accompanied by a blue shift in the intrinsic fluorescence of the protein. Single turnover kinetics for the aminoacylation of tRNA Tyr by D-tyrosine were monitored using stopped-flow fluorescence spectroscopy. Bacillus stearothermophilus tyrosyl-tRNA synthetase binds D-tyrosine with an 8.5-fold lower affinity than that of L-tyrosine (K d D-Tyr ؍ 102 M) and exhibits a 3-fold decrease in the forward rate constant for the activation reaction (k 3 D-Tyr ؍ 13 s ؊1). Furthermore, as is the case for L-tyrosine, tyrosyl-tRNA synthetase exhibits "half-of-the-sites" reactivity with respect to the binding and activation of D-tyrosine. Surprisingly, pyrophosphate binds to the TyrRS⅐D-Tyr-AMP intermediate with a 14-fold higher affinity than it binds to the TyrRS⅐L-Tyr-AMP intermediate (K d PPi ؍ 0.043 for TyrRS⅐D-Tyr-AMP⅐PP i). tRNA Tyr binds with a slightly (2.3-fold) lower affinity to the TyrRS⅐D-Tyr-AMP intermediate than it does to the TyrRS⅐L-Tyr-AMP intermediate. The observation that the K d Tyr and k 3 values are similar for Land D-tyrosine suggests that their side chains bind to tyrosyl-tRNA synthetase in similar orientations and that at least one of the carboxylate oxygen atoms in D-tyrosine is properly positioned for attack on the ␣-phosphate of ATP.
J Mol Biol, 2000
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 tRNA Tyr 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 tRNA Tyr. 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 3 H 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.
Correlating amino acid conservation with function in tyrosyl-tRNA synthetase
Journal of Molecular Biology, 2000
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 tRNA Tyr 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 tRNA Tyr. 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 3 H 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.
Amino acid activation in crystalline tyrosyl-tRNA synthetase from Bacillus stearothermophilus
Journal of Molecular Biology, 1981
A cl:\-staliinr c*omplex of tyrosyl adenylate wit,h tyrosyl-tRS.4 synthetase from Ikwillus .stmrothrrmo)-)hi/Its was prepared by the catalytic action of the crystalline enzymr. on soaking with satnrat,ed tyrosine, followed by an excess of L4TP. Difference Fourier analysis shows that tyrosyl adenylat,e takes up a conformation similar to that previously observed for the inhibitor tyrosinyl adenylate (Monteilhet R-Blow. 1978). The tyrosyl adenylat,e density straddles the central /!sheet of the structure, the adenosine and tyrosme moieties lying on opposit,e sides of the sheet. Formation of the tprosyl adenylate complex is accompanied by extensive changes in enzyme structure, which are not observed in the tyrosinyl adenplate complex. The trinucleoside diphosphate CpCpA, soaked into crystals at the same time as ATP. did not appear to bind at any localized site. IVhtw puromycin is soaked int,o tyrosyl-tRS?i synthetase crystals, strong positivr clcrtron drnsitv differences are found in the tyrosine binding site, but the changes elsewhere are weak and disconnected. Several small oligonucleotides were soaked int,o crystals and studied in projection. All indirat,e binding at, or near, the tyrosinr binding site. Difference density for arsenate substitution of the mother licluor \\'as also calcrdat,ed. showing peaks of density in the binding site and over thtx sllrfac~c~ of the molewlf~.
Protein Engineering of Tyrosyl-tRNA Synthetase: The Charging of tRNA [and Discussion]
Phil. Trans. R. Soc. Lond. A: Mathematical, Physical and Engineering Sciences, 1986
Protein engineering has been used to identify residues of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus that are in contact with the tRNA Tyr . By using improved techniques in oligonucleotide-directed mutagenesis, forty lysine, arginine, or histidine residues on the surface of the enzyme were altered to either asparagine or glutamine. With an in vivo genetic complementation test, only thirteen mutants were found that seriously affect the overall activity of the enzyme. Detailed kinetics on the purified enzymes revealed that four of these mutants had a lesion at the level of the activation of tyrosine, and nine at the level of tRNA charging. Three of the mutants in tRNA charging lie in the N-terminal domain of the enzyme which is responsible for tyrosine activation, and the six others in the disordered C-terminal domain which is necessary for tRNA binding. This indicates that the tRNA spans both domains of the enzyme. The construction of heterodimers allows us to suggest a model for tRNA binding in which the acceptor stem of the tRNA binds to the N-terminal domain of one subunit, and other regions of tRNATyr such as the anticodon arm or extra loop bind to the C-terminal domain of the other subunit.
The Aminoacyl-tRNA Synthetases - Landes Bioscience, 2005
Tyrosyl-tRNA synthetase (TyrRS) comprises an N-terminal domain, which has the fold of the class I aminoacyl-tRNA synthetases, followed by idiosynchratic domains, which differ in eubacteria, archaebacteria and eukaryotes. The eubacterial TyrRSs have recruited an RNA binding domain which is found in a large family of proteins. The crystal structures of the TyrRSs from Bacillus stearothermophilus (Bst-TyrRS) and Thermus thermophilus (Tth-TyrRS) have been solved, free, or in complex with tyrosine, or with tyrosyl-adenylate (Tyr-AMP). A quaternary complex between Tth-TyrRS, tRNATyr , tyrosinol and ATP has been solved at 2.8 Å resolution. The dimer of Bst-TyrRS is symmetrical in the crystals but asymmetrical in solution. It unfolds through a folded compact monomeric intermediate, by dissociation of the subunits (KD = 84 pM). A C-terminal domain is loosely linked to an intermediate α -helical domain through a fully flexible peptide. The tRNA binding site straddles the two subunits of TyrRS, which interacts with tRNATyr according to a class II mode. The conserved sequences of class I, HIGH and KMSKS, are involved in the catalysis of tyrosine activation. The HIGH sequence is not involved in the transfer of tyrosine from Tyr-AMP to tRNATyr, and the KMSKS sequence is involved in this transfer only through the initial binding of tRNATyr. Other residues (Thr40, Lys82 and Arg86 in Bst-TyrRS), are involved in both steps of the catalytic reaction, by interacting first with ATP then with residue Ade76 of tRNATyr. The identity elements of tRNATyr comprise nucleotidic base Ade73, the anticodon, and either base-pair Gua1:Cyt72 in eubacteria or Cyt1:Gua72 in archaebacteria and eukaryotes. The residues of TyrRS which interact with tRNATyr or recognize its identity elements have been identified by extensive mutagenesis and kinetic studies of Bst-TyrRS and from the structure of the Tth-TyrRS·tRNATyr complex. The two approaches are in excellent agreement. TyrRS catalyses the activation of tyrosine and its transfer to tRNATyr by stabilizing the transition states for these two reaction steps, through interactions with ATP, Ade76, and the identity elements of tRNATyr. The role of base pair 1:72 in the recognition of tRNATyr results in a species specificity and makes TyrRS a potential target for antibiotics. This specificity relies on a short segment (<41 residues) of TyrRS and can be swapped between species. The specific recognition between TyrRS and tRNATyr depends on the correct balance between the cellular concentrations of synthetases and tRNAs. Moreover, a residue (Glu152) of Bst-TyrRS is involved in the rejection of noncognate tRNAs but not in the interaction with tRNATyr, and thus is a purely negative determinant of specificity. Inhibitors of TyrRS have been discovered and characterized: tyrosinol, tyrosinyl-adenylate and tyrosyl-aryl dipeptides (e.g., Tyr-Tyr); however, they cross the bacterial envelope very inefficiently. Tyr-Gly dipeptides, derivatized with a sugar, have been isolated from microorganisms or synthesized, and shown to inhibit bacterial growth. The specificity of TyrRS towards the amino acid has been modified by screening or selecting mutants from random libraries which were targeted to residues of the tyrosine binding pocket. The species specificity of TyrRS towards tRNATyr and the absence of an editing mechanism towards the amino acid, have made it possible to create a 21st triplet of synthetase, tRNA and amino acid, and thus to extend the genetic code. TyrRS has additional functions in some organisms: it charges plant viral RNAs; acts as a kinase or a cytokine; plays a role in the splicing of the group I introns (as an RNA chaperone), in the maintenance of the mitochondrial genome, in yeast sporulation and in the quality control of tRNAs in the cell nucleus. TyrRS has been used in the synthesis of analgesic neuro-dipeptides.