Thermodynamic Analysis Reveals a Temperature-dependent Change in the Catalytic Mechanism of Bacillus stearothermophilus Tyrosyl-tRNA Synthetase (original) (raw)
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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
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.
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.
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.
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~.