Distal mutations in a designed retro-aldolase alter loop dynamics to shift and accelerate the rate-limiting step

Amino-acid residues distant from an enzyme’s active site are known to influence catalysis, but their mechanistic contributions to the catalytic cycle remain poorly understood. Here, we investigate the structural, functional, and mechanistic impacts of distal and active-site mutations discovered through directed evolution of the computationally designed retro-aldolase RA95. Active-site mutations improve catalytic efficiency by 3,600-fold, while distal mutations alone offer no improvement. When combined with active-site mutations, distal mutations further increase efficiency by 6-fold, demonstrating an epistatic effect. X-ray crystallography and molecular dynamics simulations reveal that distal mutations promote active site opening by altering loop dynamics. Kinetic solvent viscosity effects and electrostatic analysis show that distal mutations accelerate the chemical transformation by 100-fold, shifting the rate-limiting step to product release, which is further accelerated by the increased opening of the active site. These findings highlight the critical role of distal residues in shaping the active-site environment and facilitating the structural dynamics essential for progression through the catalytic cycle.