To further explore the impact of the binding of the two substrates on loop dynamics, we extended our simulations to also included simulations of TrpF in both its unliganded form, and in complex with PRA (Table ). We then performed Principal Component Analysis (PCA) to characterize the motion of the key catalytic loops during our simulations in each of the individual systems, similarly to prior analysis we have performed on triosephosphate isomerase, except in our prior work substantial conformational changes take place in only one and not two (TmTrpF) or three (SeHisA; MtPriA) distinct loops. The PCA analysis was performed on the mass-weighted Cartesian coordinates of each enzyme compared to the coordinates of the corresponding closed state, allowing us to explore the variation of the conformations of these loops in coordinate space. We subsequently projected the free energies for each enzyme along the most dominant motions, PC1 and PC2, from simulations of each of HisA, PriA and TrpF in their unliganded forms as well as in complex with substrates ProFAR and PRA, respectively (here, the smaller substrate PRA was artificially placed in the HisA active site by manual overlay with the reactive part of ProFAR, as outlined in the Supporting Information). This allowed us to compare the free energy surfaces defined by these two principal components both between the different enzymes, and the effect of ligand binding on these surfaces. The resulting data are shown in Figure . Note that, as shown in Figure , these projected free energy surfaces show the combined motion of all key catalytic loops along each principal component. In the unliganded forms of all three enzymes, the catalytic loops can explore a range of "wideopen" conformations and are overall highly conformationally diverse (Figure ). This observation is consistent with our prior simulation studies on both triosephosphate isomerase, and the protein tyrosine phosphatases PTP1B and YopH, as well as chimeric forms of these enzymes. However, and consistently with structural data, the binding of ProFAR to HisA fully restricts the conformational sampling of all three loops (Figure ). In the case of PriA, the binding of both ProFAR and PRA also stabilizes the closed conformation of the three active site loops, but still allows for some conformational flexibility in these loops (Figure , based on both the topologies of the projected free energy surfaces, and the corresponding energies). In sharp contrast, in the liganded form of TrpF, our MD simulations show that PRA is not stable in the active site due to the flexibility of loop 6, which explores transitions towards open conformations, similarly to the unliganded system (Figures and). In the crystal structure of the unliganded enzyme (PDB ID: 1NSJ ), this loop is present in a closed position but with missing density, whereas in our simulations of both the liganded and unliganded form of the enzyme, loop 6 samples open conformations, suggesting that it is not the correct closed state to stabilize the substrate PRA in the active site. We note that the structure used for these simulations (PDB ID 1LBM ) was solved in complex with the product analog rCdRP. Our simulations suggest that the loop conformations observed in this structure are a conformational state on the trajectory to product release, rather than an ideal conformational state for stabilizing the Michaelis complex.