Figure 3.
A) Protection with ADP against DFTD. eEF-2K was incubated in the presence of ADP (4 mM) and/or DFTD (250 μM) for different timepoints (0, 5, 10, 15, 20, and 30 min). The enzyme-inhibitor mixtures were diluted 50 fold into the assay reaction. The formation of E•I* complex in the presence (■) or absence (●) of ADP were plotted against different incubation times. Data were fitted using Equation 1, where the kobs for the formation of the E•I* complex in the presence and absence of ADP are 0.0002 ± 0.00003 and 0.0015 ± 0.0003 s−1. B) Recovery of enzyme activity against DFTD for different mutants (◆,C146A; ■, C146S; ▲, Y236F; ●, wildtype). Wildtype and mutant eEF-2K were first incubated with DFTD (1 mM) for 60 min, diluted 50 fold, and incubated for different timepoints before starting the assay. The recovery of enzyme activity upon dilution over time were fitted using Equation 1.