Analysis of the steady-state mechanism of the aminoacylation of tRNAPhe by phenylalanyl-tRNA synthetase from yeast.

The steady-state mechanism of the aminoacylation of tRNAPhe by the corresponding synthetase from yeast has been investigated in detail by kinetic experiments. It was found that there are two alternative mechanisms: one favoured at low tRNA concentrations and the other at high tRNA concentrations. ATP and Phe are bound randomly to the enzyme. AMP is released immediately after the binding of ATP and Phe. Between the release of AMP and pyrophosphate (PPi) there is at least one additional step. Based on the experimental results a model of the steady-state mechanism is proposed. This model includes the sequence of addition of substrates to the enzyme and the release of products from the enzyme as well as the composition of the intermediate complexes with the enzyme. This model is in accordance with previous results based on different techniques. The results are explained by a "flip-flop" mechanism for all the substrates and products involved in the reaction.