The empirical valence bond (EVB) approach has been extensively used to successfully study enzyme catalysis in general, and Kemp elimination in particular. In this context, we recently used the EVB approach to study the evolution of multiple active site configurations in the de novo designed Kemp eliminase, KE07. In the present work, we follow the protocol presented in ref. . Our EVB simulations were performed using a simple two-state EVB model, describing the reactant and product states for the Kemp elimination reaction, with the side chain of D229 and the substrate included in the EVB region. All other residues were treated fully classically using the OPLS-AA force field. All simulations were performed using the Q simulation package, version 5.10, and a description of valence bond states and all EVB parameters used in the simulations are provided in the Supporting Information of ref. . EVB simulations were performed of the Kemp elimination reaction catalyzed by the GNCA4-WT β-lactamase, a series of additional single active site mutations of this variant used for calibration of the EVB simulations (G62S, A146G, A173V, L265Q, R256K, R256A), as well as the top-twenty ranked mutations predicted by the FuncLib web-server, based on both the structural predictions from FuncLib, and, where available, also crystal structures for comparison (for the three variants characterized in this work). Simulations of the GNCA4-WT variant were performed using the PDB ID: 5FQK, and the best hits from the FuncLib webserver were simulated based on the PDB structures provided by FuncLib 28 with the substrate. The structures of all other variants were generated using SCWRL4. In all cases, the substrate 5-nitrobenzisoxazole was manually placed in the active site in the position of the transition state analogue 5(6)-nitrobenzotriazole present in the crystal structure. Missing residues at the C-and N-termini of the protein were ignored for simplicity, and the first residue of the His-tag present in the initial crystal structure was retained for consistency (this was also the case for the FuncLib calculations).