Investigating the Non-Electrostatic Component of Substrate Positioning Dynamics

Substrate positioning dynamics (SPD), which orients the substrate to a reactive conformation in the active site, is critical in mediating enzyme catalysis. However, given that conformational changes often accompany variations in the enzyme interior electrostatics, it remains unknown whether SPD contains a non-electrostatic component that independently mediates catalysis, or originates primarily from perturbation of enzyme interior electrostatics. This study integrated computational and experimental approaches to investigate the non-electrostatic component of SPD using Kemp eliminase (KE) as a model enzyme. A molecular dynamics-derived descriptor, substrate positioning index (SPI), was used to quantify the impact of protein dynamics on substrate positioning. Using high throughput enzyme modeling, we selected 7 KE variants for kinetic assessment – these variants involved significantly different SPD but similar interior enzyme electrostatics. We observed a valley-shaped, two-segment piecewise linear correlation between the experimentally characterized activation free energies and SPI values. The trend is further validated using previously reported kinetic data. An optimal SPI value, corresponding to the lowest activation free energy, was observed for R154W, a surface mutation located distantly from the active site. Compared to the wild type, R154W involves favorable SPD that increases the proportion of reactive conformations for substrate deprotonation. These results indicate the presence of the non-electrostatic component of SPD, a concrete factor that mediates catalysis by tuning the population of reactive conformation.