Thermodynamic Analysis Reveals a Temperature-dependent Change in the Catalytic Mechanism of Bacillus stearothermophilus Tyrosyl-tRNA Synthetase

Catalysis of tRNATyr 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 tRNATyr 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 °C, where the values of ΔH0 and ΔS0 go from –144 kJ/mol and –438 J/mol K below 25 °C to +137.9 kJ/mol and +507 J/mol K above 25 °C. Nonlinear Eyring and van't Hoff plots are also observed for formation of the TyrRS·[Tyr-ATP]‡ 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 ΔH0 and ΔS0 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 ΔH0 and ΔS0 are because of the KMSKS signature sequence being conformationally constrained and unable to disrupt this synergistic interaction below 25 °C.