Investigating the Non-Electrostatic Component of Substrate Positioning Dynamics
Substrate positioning dynamics (SPD) orients the substrate to reactive conformations in the active site, accelerating enzymatic reactions. However, it remains unknown whether SPD effects originate primarily from electrostatic perturbation inside the enzyme or can independently mediate catalysis with a significant non-electrostatic component. Here we investigated how the non-electrostatic component of SPD affects transition state stabilization. Using high-throughput enzyme modeling, we selected Kemp eliminase variants with similar electrostatics inside the enzyme but significantly different SPD. The kinetic parameters of these selected mutants were experimentally characterized. We observed a valley-shaped, two-segment linear correlation between the TS stabilization free energy (converted from kinetic parameters) and an index used to quantify SPD. Favorable SPD was observed for a distal mutant R154W, leading to the lowest activation free energy among the mutants tested. R154W involves an increased proportion of reactive conformations. These results indicate the contribution of the non-electrostatic component of SPD to mediating enzyme catalytic efficiency.