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

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.
Mesh Terms:
Amino Acyl-tRNA Synthetases, Aspartic Acid, Binding Sites, Enzyme Activation, Humans, Hydrolysis, Kinetics, Peptide Elongation Factor 1, Peptide Elongation Factors, RNA, Transfer, Amino Acyl
J. Biol. Chem.
Date: Dec. 30, 1994
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