The mechanism of the aminoacylation of transfer ribonucleic acid: enzyme-product dissociation is not rate limiting.
It has been proposed that the rate-limiting step in the synthesis of aminoacyl-tRNA is the rate at which the product dissociates from the enzyme. The experimental evidence supporting this hypothesis comes from work at low pH and low temperature (although the reaction has been argued to have the same mechanism under physiological conditions). We have reexamined the binding assay by which M. Yarus and P. Berg (1969) (J. Mol. Biol. 42, 171-189) measured the kd for dissociation of Enz-(Ile-tRNA). We find that when overall reaction and dissociation are measured under identical conditions the two rates are not the same. Moreover, while an increase in ionic strength greatly stimulates dissociation, the same increased ionic strength slows aminoacylation. Spermine accelerates overall aminoacylation without affecting dissociation. Because any change in a rate-limiting step must, by definition, cause a parallel change in the overall reaction, these observations prove that under these conditions the synthesis of Ile-tRNA is not limited by the rate of dissociation of Enz-(Ile-tRNA). Entirely similar observations were made for the dissociation of Enz-(Val-tRNA) and the overall synthesis of Val-tRNA at 0 degrees C, PH 5.0. In addition, valine enzyme isolated by nitrocellulose filtration during the course of an aminoacylation was shown not to be saturated with recently synthesized Val-tRNA. The enzyme was in equilibrium with uncharged substrate tRNA and with product Val-tRNA. E. W. Eldred and P. R. Schimmel ((1972) Biochemistry 11, 17-23) report that the formation of Ile-tRNA proceeds at two rates: (a) k = 2 X 10(-2)S(-1) until the enzyme is saturated with the first mole of product, and (b) k = 2 X 10(-3)S(-1) for subsequent cycles. We did not observe this behavior at any pH or temperature with four different amino acid:tRNA ligases. Because aminoacylation proceeds more rapidly than "dissociation" under some conditions, we believe that the binding assay measures not only enzyme-product dissociation but also other slower reactions such as aggregation or disaggregation of Enz-(AA-tRNA). In conjunction with recent studies from other laboratories, this work makes it unlikely that enzyme-product dissociation is the rate-limiting step in the synthesis of aminoacyl-tRNA either at low temperature and pH or under more nearly physiological conditions. From the effect of salt, it would appear that the rate of aminoacylation of tRNA is largely limited by the rate or extent of formation of Enz-(tRNA) (Loftfield, R. B., and Eigner, E. A. (1967), J. Biol. Chem. 242, 5355-5359). Using the binding assay of M. Yarus ((1972) Biochemistry 11, 2050-2060), we find the Kass for Enz-(Ile-tRNA) varies linearly with the Debye-Hückel function at ionic strengths of 0.1-0.4 from 10(8) to 10(6).
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D011485:Protein Binding; D012343:RNA, Transfer; D013997:Time Factors
D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase