Fig. (3). Time course of inactivation of IleRS by tRNAIleox.
IleRS (1 μM) was incubated with 4 μM tRNAIleox, as described under Methods. The residual isoleucine-dependent isotopic [32P]PPi-ATP exchange (●, ▲, ❍) and tRNAIle aminoacylation (■, ◆, ❐)activities were measured as a function of time. The control experiments (❍ , ❐) were without tRNAIleox or with 4 µM intact tRNAIle instead of tRNAIleox. For the protection experiments (▲ , ◆), 20 µM of intact tRNAIle was present with the enzyme prior to tRNAIleox addition. In another protection experiment, IleRS (1 µM) was pre-incubated with a combination of 8 mM MgATP and 8 mM L-isoleucine, prior to tRNAIleox addition. In the latter case, kinetics of enzyme activity loss for isoleucine-dependent isotopic [32P]PPi-ATP exchange and tRNAIle aminoacylation activities were superimposable on those of the controls (❍ , ❐).