Abstract
Catalysis of tRNA(Tyr) aminoacylation by tyrosyl-tRNA synthetase can be divided into two steps. In the first step, tyrosine is activated by ATP to form the tyrosyl-adenylate intermediate. In the second step, the tyrosyl moiety is transferred to the 3' end of tRNA. To investigate the roles that enthalpic and entropic contributions play in catalysis by Bacillus stearothermophilus tyrosyl-tRNA synthetase (TyrRS), the temperature dependence for the activation of tyrosine and subsequent transfer to tRNA(Tyr) has been determined using single turnover kinetic methods. A van't Hoff plot for binding of ATP to the TyrRS.Tyr complex reveals three distinct regions. Particularly striking is the change occurring at 25 degrees C, where the values of DeltaH(0) and DeltaS(0) go from -144 kJ/mol and -438 J/mol K below 25 degrees C to +137.9 kJ/mol and +507 J/mol K above 25 degrees C. Nonlinear Eyring and van't Hoff plots are also observed for formation of the TyrRS.[Tyr-ATP](double dagger) and TyrRS.Tyr-AMP complexes. Comparing the van't Hoff plots for the binding of ATP to tyrosyl-tRNA synthetase in the absence and presence of saturating tyrosine concentrations indicates that the temperature-dependent changes in DeltaH(0) and DeltaS(0) for the binding of ATP only occur when tyrosine is bound to the enzyme. Previous investigations revealed a similar synergistic interaction between the tyrosine and ATP substrates when the "KMSKS" signature sequence is deleted or replaced by a nonfunctional sequence. We propose that the temperature-dependent changes in DeltaH(0) and DeltaS(0) are because of the KMSKS signature sequence being conformationally constrained and unable to disrupt this synergistic interaction below 25 degrees C.
Highlights
AaRS 1⁄7 AA-AMP ϩ tRNAAA ^ aaRS ϩ AA-tRNAAA ϩ AMP REACTION 2 where aaRS,2 AA, and tRNAAA represent the aminoacyl-tRNA synthetase, its amino acid substrate, and the cognate tRNA, respectively, and “1⁄7” and “Ϫ” represent noncovalent and covalent interactions, respectively
We investigate the role that enthalpy and entropy play in catalysis of tRNATyr aminoacylation by B. stearothermophilus tyrosyl-tRNA synthetase using single turnover conditions
Despite the nonlinearity observed in the van’t Hoff and Eyring plots, the Arrhenius plot is linear for the single turnover kinetic data. This observation indicates that the nonlinearity observed for the binding of ATP (Fig. 2, panel B) is entirely offset by the nonlinearity observed in k3, the forward rate constant (Fig. 2, panel C). This suggests that the nonlinearity observed for formation of the TyrRS1⁄7Tyr1⁄7ATP and TyrRS1⁄7[TyrATP]‡ complexes has the same physical basis and is consistent with the hypothesis that the KMSKS sequence reduces the synergistic interaction between tyrosine and ATP on the initial binding of ATP, allowing it to instead be used to stabilize the TyrRS1⁄7[Tyr-ATP]‡ transition state
Summary
Materials—Reagents were purchased from the following sources: DE52 anion-exchange resin, Whatman; -mercaptoethanol, disodium pyrophosphate, and inorganic pyrophosphatase, Sigma; nitrocellulose filters, Schleicher & Schuell; Source 15Q-Sepharose and NAP-25 columns, Amersham Biosciences. The binding of ATP to tyrosyl-tRNA synthetase does not induce a change in the intrinsic fluorescence of the enzyme, the equilibrium constants for the dissociation of ATP from the TyrRS1⁄7Tyr1⁄7ATP and TyrRS1⁄7ATP complexes can be determined kinetically This is done by monitoring formation of the TyrRS1⁄7Tyr-AMP intermediate under conditions where the enzyme is initially present as either the TyrRS1⁄7Tyr complex The equilibrium constant for the dissociation of ATP from the TyrRS1⁄7ATP complex (KdATP) is calculated from the variation of kobs with respect to the ATP concentration in the presence of either 0.1 or 1.0 M tyrosine (similar values for KdATP were obtained for both concentrations of tyrosine) Under these conditions, at least 90% of the tyrosyl-tRNA synthetase is present as the free enzyme [49]. To compare the thermodynamic values obtained using single turnover kinetics with previously published thermodynamic values obtained using steady state (multiple turnover) kinetic methods, a plot shown in Equation 11
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