Here, we have simulated both Chimeras 2 and 3 from as well as wild-type YopH, starting from a different conformations of the WPD-loop, and combining conventional and targeted molecular dynamics simulations with EVB simulations of the rate-limiting hydrolysis step. As in our prior work , EVB simulations initiated from idealized closed conformations suggest negligible energetic differences between the different enzyme variants, which is to be expected as these enzymes have essentially identical active site architectures that are not modified by the alterations in WPD-loop sequence. This points towards a putative dynamical origin for the differences in activity. Our conventional and targeted molecular dynamics simulations indicate significant differences in the stability of the wide-open conformation of the WPD-loop, which is more stable in inactive Chimera 2 than in active Chimera 3 or wild-type YopH, suggesting that it is more challenging for Chimera 2 to sample a catalytically active conformation.