The mechanism of the aminoacylation of transfer ribonucleic acid: enzyme-product dissociation is not rate limiting

Abstract

Enzyme[Enz]-product dissociation was proposed as the rate-limiting step in the synthesis of aminoacyl-tRNA. Support for this hypothesis comes from work at low pH and low temperature. The reaction may have the same mechanism under physiological conditions. A technique for measuring the Kd of Enz.cntdot.(Ile-tRNA) was examined. When overall reaction and dissociation are measured under identical conditions the 2 rates are not the same. An increase in ionic strength greatly stimulates dissociation, but slows aminoacylation. Spermine accelerates overall aminoacylation without affecting dissociation. Since any change in a rate-limiting step must 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 dissociation rate of Enz.cntdot.(Ile-tRNA). Entirely similar observations were made for Enz.cntdot.(Val-tRNA) dissociation and the overall synthesis of Val-tRNA at 0.degree. C, pH 5.0. In addition, valine enzyme isolated by nitrocellulose filtration during the course of an aminoacylation was not saturated with recently synthesized Val-tRNA. The enzyme was in equilibrium with uncharged substrate tRNA and with product Val-tRNA. The formation of Ile-tRNA proceeding at 2 rates k = 2 .times. 10-2s-1 until the enzyme is saturated with the first mole of product, and k = 2 .times. 10-3s-1 for subsequent cycles, was not observed at any Ph or temperature with 4 different amino acid:tRNA ligases. Because aminoacylation preceeds more rapidly than dissociation under some conditions, the binding assay may measure enzyme.cntdot.product dissociation plus other slower reactions such as aggregation or disaggregation of Enz.cntdot.(AA-tRNA). This work makes it unlikely that enzyme.cntdot.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.cntdot.(tRNA). The binding constant for Enz.cntdot.(Ile-tRna) varies linearly from 108-106 with the Debye-Huckel function at ionic strengths of 0.1-0.4.