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 electric
field calculations 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 establish the critical role of distal
residues in shaping the active-site environment and facilitating the
structural dynamics essential for efficient progression through the
catalytic cycle, offering valuable insights for enzyme design.