Conformation change of tRNAGlu in the complex with glutamyl-tRNA synthetase is required for the specific binding of L-glutamate.
The binding of Thermus thermophilus glutamyl-tRNA synthetase (GluRS) with T. thermophilus tRNAGlu, Escherichia coli tRNAGlu, and amino acids was studied by fluorescence measurements. In the absence of tRNAGlu, GluRS binds with D-glutamate as well as L-glutamate. However, in the presence of E. coli tRNAGlu, GluRS binds specifically with L-glutamate. The KCl effects on the Michaelis constants (Km) for tRNAGlu, L-glutamate, and ATP were studied for the aminoacylation of the homologous tRNAGlu and heterologous tRNAGlu species. As the KCl concentration is raised from 0 to 100 mM, the Km value for L-glutamate in the heterologous system is remarkably increased whereas the Km value for L-glutamate in the homologous system is only slightly increased. The circular dichroism analyses were made mainly of the bands due to the 2-thiouridine derivatives of tRNAGlu in the complex. The conformation change of T. thermophilus tRNAGlu upon complex formation with GluRS is not affected by addition of KCl. In contrast, the heterologous tRNAGlu X GluRS complex is in an equilibrium of two forms that depends on KCl concentration. The predominant form at low KCl concentration is closely related to the small Km value for L-glutamate. In this form of the complex, the conformation of tRNAGlu is appreciably different from that of free molecule. Accordingly, such a conformation change of tRNAGlu in the complex with GluRS is required for the specific binding of L-glutamate as the substrate.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005975:Glutamate-tRNA Ligase / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D007700:Kinetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000302:isolation & purification / Q000378:metabolism*; D013824:Thermus / Q000201:enzymology*
D005971:Glutamates; D012346:RNA, Transfer, Amino Acyl; D018698:Glutamic Acid; D000604:Amino Acyl-tRNA Synthetases; D005975:Glutamate-tRNA Ligase