BPS2026 – Who's really in charge? Dissecting directional electric fields in enzyme catalysis

Electrostatic preorganization is a driver of enzyme catalysis, yet the directional components of electric fields and their roles in catalysis remain poorly understood. To probe the role of directional fields, we employed a deuterated hydride on 4R- 2 H-NADH as a vibrational Stark probe in horse liver alcohol dehydrogenase (LADH), complexed with a deuterated aldehyde substrate analog, cyclohexaformamide (CXF-D). This approach enabled simultaneous measurement of electric fields along three critical vectors: the hydride transfer axis, the carbonyl bond of CXF-D, and the orthogonal C-D bond on the aldehyde of CXF-D. These measurements reveal catalytic preorganization in the LADH active site, extending beyond the substrate carbonyl fields. Crystallographic, spectroscopic, and kinetic analyses of variants such as Thr178Ser revealed that removing the Thr methyl group disrupts cofactor positioning and increases conformational heterogeneity—effects that occur independently of the local electric field experienced by the Stark probes along the carbonyl and aldehyde of CXF-D. This is confirmed by IR measurements showing two non-interconverting populations in LADH mutants where the methyl group is present (Thr178, Val178), and a single population where it is absent (Ala178, Ser178)—without changes in fields on the carbonyl or aldehyde probes, supported by electron density differences observed in high-resolution crystal structures, and all-atom MD simulations. Double mutant analysis with Ser48—a residue that generates a strong catalytic field via hydrogen bonding to the carbonyl—reveals complementary but distinct effects on activity. While packing interactions involving Thr178 modulate catalysis, their contribution is an order of magnitude smaller than that of electrostatic fields. These findings identify Thr178 as a modulator of ground-state destabilization and demonstrate how local structure and electrostatics cooperatively shape the catalytic landscape. This work establishes a framework for probing directional electric fields in enzymes and guiding the design of electrostatically optimized biocatalysts.