The performance of Q-RepEx against conventional EVB simulations was tested on three reactions that we have studied extensively in prior work: the isomerization of substrate Dglyceraldehyde-3-phosphate (GAP) by triosephosphate isomerase from Saccharomyces cerevisiae (yTPI) , the isomerization of substrate ProFAR by PriA from Mycobacterium tuberculosis (MtPriA) , and the rate-limiting hydrolysis of the phosphoenzyme intermediate formed during the reaction catalyzed by protein tyrosine phosphatase 1B (PTP1B) . Here, we used the same system setup and starting structures as in prior studies for all three systems (PDB IDs 1NEY , 3ZS4 and 3I80 for yTPI, MtPriA and PTP1B, respectively), as well as the EVB equilibration procedure outlined in detail in Ref. . In the case of MtPriA, for simplicity, we selected 10 equilibration trajectories out of the 30 total generated for our prior work (selecting every third trajectory we ran) , and used these equilibration endpoints as the starting points for our various Q-RepEx simulations. As PriA is a bi-functional enzyme, we used here PDB ID: 3ZS4 , which has loop 5 of PriA in a "pro-ProFAR" conformation necessary for catalyzing the substrate studied here (see discussion in e.g. refs. ). In the case of yTPI and PTP1B , as there were slight differences in the equilibration procedure used for those works and our MtPriA study , we generated 10 new equilibrated trajectories using the protocol described for PriA in Ref. , and used these as starting points for our various REMD-EVB calculations, using PDB ID: 1NEY (Saccharomyces cerevisiae TPI in complex with substrate dihydroxyacetone phosphate) as our starting point. The root mean square deviations (RMSD) of all backbone atoms during our respective equilibration runs is shown in Figure .