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.
Reed
V S
VS
Department of Chemistry, Georgetown University, Washington, D.C. 20057.
Wastney
M E
ME
Yang
D C
DC
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*
D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; D001224:Aspartic Acid; D000604:Amino Acyl-tRNA Synthetases