Quantifying and Engineering Electric Fields in Liver Alcohol Dehydrogenase.

Electrostatic interactions play a central role in enzyme catalysis by stabilizing charge redistribution along the reaction coordinate. In liver alcohol dehydrogenase (LADH), experimental measurements using vibrational probes interpreted through Vibrational Stark Effect (VSE) spectroscopy have demonstrated a strong correlation between the electric field projected along a bound carbonyl probe and catalytic rate across metal substitutions and active-site mutations. Here, we employ Quantum Mechanics/Molecular Mechanics (QM/MM) calculations to quantify and predict the electric fields within LADH and to evaluate their modulation by targeted mutations. The computed electric fields reproduce the experimentally observed trends in projected field magnitudes. By systematically analyzing how specific residue substitutions perturb the projected electric field along the C═O bond of a probe, we establish a quantitative computational framework for predicting electrostatic perturbations in LADH, consistent with experimentally observed catalytic trends. This work advances the integration of QM/MM electrostatics with experimentally measurable electric-field observables in enzyme systems.