Loop closure over the active site elevates the pKa of a key catalytic glutamic acid (estimated to be as high as ~10 in the presence of trianionic phosphoglycolate ), which, in turn, allows this side chain to deprotonate the substrate. This initial proton transfer step is expected to be rate-limiting, with subsequent steps in the isomerization to be fast in comparison to the initial proton transfer, and thus the initial substrate deprotonation has been the focus of our prior EVB simulations of wild-type and variant forms of TPIs from a range of different organisms, with good agreement with experiment . Here, we use the initial deprotonation of substrate DHAP as catalyzed by wild-type TPI from Saccharomyces cerevisiae (yTPI) as a model reaction for evaluating the performance of Q-RepEx.