Fig. (3) Time course of inactivation of IleRS by tRNA^Ileox. IleRS (1 μM) was incubated with 4 μM tRNA^Ileox, as described under Methods. The residual isoleucine-dependent isotopic [³²P]PPi-ATP exchange (●, ▲ ○) and tRNA^Ile aminoacylation (■ ◆, ◻) activities were measured as a function of time. The control experiments (○ , □) were without tRNA^Ileox or with 4 µM intact tRNA^Ile instead of tRNA^Ileox. For the protection experiments (▲ ◆), 20 µM of intact tRNA^Ile was present with the enzyme prior to tRNA^Ileox 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 tRNA^Ileox addition. In the latter case, kinetics of enzyme activity loss for isoleucine-dependent isotopic [³²P]PPi-ATP exchange and tRNA^Ile aminoacylation activities were superimposable on those of the controls (○ , □).