In Search of Rapid Dynamics: How Fast Protein Motions Influence Enzymatic Catalysis

Abstract

Understanding how rapid, femtosecond-level protein dynamics enhance the catalytic rate of an enzyme is challenging due to the range of timescales. While it is widely accepted that protein motions have specific functions in enzymes, there is debate over whether the dynamics during the chemical step are coupled to catalysis. A hierarchy of timescales within protein motions exists, affecting stability, substrate affinity, product release, and chemistry. Studies into protein dynamics significantly impact protein engineering, particularly in artificial enzyme design. Currently, these artificial enzymes produce low catalytic rates compared to natural enzymes, except in certain cases generated through directed evolution. By analyzing the coupling between rapid dynamics and slow conformational motions, and identifying dynamics maintained throughout evolution, we can gain insights into the necessary modifications in terms of rapid dynamics to produce efficient artificial enzymes. Conformational motions, which are much slower relative to femtosecond-level motions, assist in catalysis, protein folding, and substrate specificity. Rapid promoting vibrations, as seen in transition path sampling studies, contribute to enzyme catalysis. To uncover the coupling of rapid promoting vibrations and conformational motions, I studied the enzyme purine nucleoside phosphorylase, confirming the connection between these two types of motions. Constraining a significant conformational motion, the loop that closes over the active site, affects the reaction coordinate, the electric field size in the active site, and the free energy barrier. Despite the vast difference in timescales, we demonstrated how altering this loop motion decreases the effects of the rapid promoting vibration. Evolutionary mutations provide insights into the role of dynamics in catalysis. Identifying mutations that improve function is essential for synthesizing enzymes that can compete with the catalytic rates of natural enzymes. Using molecular dynamics simulations and Transition Path Sampling methods, we identified rate-enhancing dynamics at the femtosecond level in both ancestral and modern enzymes. We found that these fast motions are more efficiently coordinated in the modern enzyme and examined how specific dynamics can pinpoint evolutionary effects essential for improving enzymatic catalysis.