Mechanisms of the transfer of aminoacyl-tRNA from aminoacyl-tRNA synthetase to the elongation factor 1 alpha

• Published in: The Journal of biological chemistry 1994-12, Vol.269 (52), p.32932-32936
• Main Authors: Reed, V S, Wastney, M E, Yang, D C
• Format: Article
• Language: English
• Subjects: Amino Acyl-tRNA Synthetases - genetics; Amino Acyl-tRNA Synthetases - metabolism; Aspartic Acid - metabolism; Binding Sites; Enzyme Activation; Humans; Hydrolysis; Kinetics; Peptide Elongation Factor 1; Peptide Elongation Factors - metabolism; RNA, Transfer, Amino Acyl - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(20)30080-6

Aspartylation of mammalian tRNAAsp by bacteria-expressed human aspartyl-tRNA synthetase (hDRS) was examined. The kinetics of the aspartylation of tRNA was consistent with the following reaction pathway, [formula: see text] where E, represents aspartyl-tRNA synthetase. A set of rate constants was obtained which fit single turnover time courses at varying concentrations of the enzyme, tRNA, and AMP using the SAAM program. The dissociation of Asp-tRNA (k3) was found to be rate limiting. The elongation factor 1 alpha (EF1 alpha) and GTP stimulated the hDRS aspartylation. The stimulation depended on the presence of both EF1 alpha and GTP. Kinetic analysis indicated that EF1 alpha formed a complex with the hDRS-Asp-tRNA complex and stimulated the dissociation of Asp-tRNA. In the presence of 0.5 M NH4Cl, which enhances the binding of Asp-tRNA by EF1 alpha, hDRS-bound Asp-tRNA can be transferred directly to EF1 alpha. The implications of these results on the function of the multi-tRNA synthetase complex will be discussed.