title: Thermodynamic analysis reveals a temperature-dependent change in the catalytic mechanism of bacillus stearothermophilus tyrosyl-tRNA synthetase.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130, USA.
authors.forename: Gyanesh
authors.initials: G
authors.lastname: Sharma
authors.forename: Eric A
authors.initials: EA
authors.lastname: First
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D011485:Protein Binding; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012365:RNA, Transfer, Tyr; D014443:Tyrosine; D000255:Adenosine Triphosphate; D014448:Tyrosine-tRNA Ligase