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.已知远离酶活性位点（active site）的氨基酸残基可影响催化作用，但其对催化循环（catalytic cycle）的机制性贡献仍有待深入阐明。本研究针对通过计算设计的逆向醛缩酶RA95（computationally designed retro-aldolase RA95）的定向进化（directed evolution）所获得的远端突变与活性位点突变，探究其结构、功能及机制层面的影响。活性位点突变可将催化效率提升3600倍，而单独的远端突变则无改善效果。当与活性位点突变联合使用时，远端突变可进一步将效率提升6倍，体现出上位性效应（epistatic effect）。X射线晶体学（X-ray crystallography）与分子动力学模拟（molecular dynamics simulations）结果显示，远端突变通过改变环区动力学特性促进活性位点开放。溶剂粘度动力学效应（kinetic solvent viscosity effects）与电场计算（electric field calculations）表明，远端突变可将化学转化速率提升100倍，将限速步骤（rate-limiting step）转变为产物释放（product release），而活性位点开放程度的提升进一步加速了该过程。本研究明确了远端残基在塑造活性位点环境、调控催化循环高效推进所必需的结构动力学方面的关键作用，可为酶设计提供极具价值的参考思路。