Dynamic Allostery in Enzyme Catalysis: A Time-Resolved Crystallography Study

Abstract

This study employs time-resolved XFEL crystallography to investigate dynamic allosteric mechanisms in enzyme catalysis. Structural states were monitored between 5-500 ms after substrate binding, capturing tetrahedral intermediate formation at 100 ms with allosteric site expansion (35% volume increase) that occurs 35 ms after active site contraction. Electrostatic perturbations propagate faster (0.54 ± 0.06 Å/ms) than volume changes (0.43 ± 0.05 Å/ms) or flexibility changes (0.36 ± 0.04 Å/ms), with very good correlation between active site chemistry and allosteric response (R² = 0.87). These findings reveal a hierarchical mechanism whereby electrostatic reorganization occurs before further conformational changes, reconciling rival models of enzyme function. This study provides a framework for the exploration of dynamic allostery in enzyme mechanisms and allows systematic design of modulators of individual steps of such dynamic processes.