Preparation and screening of saturation libraries. Saturation libraries at the positions in the active-site set were prepared using the QuickChange Lighting PCR method (Agilent #210518). The codon corresponding to each position was replaced by NNN. Library preparation, transformation and quality control of the library were carried as we have previously described in detail. Library screening was also performed following exactly a protocol that we recently described and that took advantage of the availability of a Freedom EVO 200 robot from TECAN (Männedorf, Schweiz), except for the buffer and the Kemp substrate concentration employed: HEPES 10 mM NaCl 100 mM pH 8.5 and 0.06 mM, respectively. About 90 colonies were screened for each library to thus ensure a high probability that all of the 20 variants were screened at least once. Results are shown in Figure . Sequence-specific assignments were made using the standard procedures on the basis of the following experiments: 2D 1 H, N HSQC and 3D HNCO and HNCA using a BEST TROSY versions. NMRPipe 63 and NMR View 64 were used to process the raw NMR data and to perform interactive spectrum analysis, respectively. Chemical shifts were referenced to the water signal as an internal reference for 1 H using pH and temperature corrections. N and 13 C chemical shifts were referenced indirectly. The titration experiments were performed by stepwise addition of a concentrated solution of the transition-state analogue to a 13 C, the GNCA4-WT, GNCA4-12, and the V4-4 variant, the latter being simulated both with and without the additional polypeptide segment. All simulations were performed in complex with the substrate 5-nitrobenzisoxazole, which was manually modelled into all systems based on the position of the transition state analogue (TSA) 6-nitrobenzotriazole in the GNCA4-WT structure (PDB ID: 5FQK 28 ). In the case of GNCA4-WT and GNCA4-12, available crystallographic structures were used as starting points for the simulation (PDB IDs: 5FQK and 6TXD, respectively. The polypeptide segment of V4 was modelled into the GNCA4-12 construct from sequence using Modeller. Finally, the V4-4 starting structure was prepared by first manually inserting the corresponding mutations into the V4 construct, followed by structure optimization using AlphaFold. In all cases, the substrate 5-nitrobenzioxazole was manually placed in the active site in the position of the transition-state analogue 5(6)-nitrobenzotriazole present in the crystal structure. Additionally, His tags were retained in our simulations for better direct comparison with experiment. All starting structures employed during this work, and any modifications made to them during setup, are summarized in Table . Descriptions of system set up for additional simulations provided in this work for KIF analysis are provided as Supporting Information.