Effect of ²H₂O solvent on protein conformational sampling linked to catalysis by UDP-glucuronic acid 4-epimerase

²H₂O solvent causes structural rigidification of proteins, but how this effect impacts enzyme catalysis is not well understood. We show for UDP-glucuronic acid 4-epimerase (UGAepi) that, compared to ¹H₂O, reaction in ²H₂O involves more negative activation heat capacity (ΔΔC_p^‡ = 1.2 kJ mol⁻¹ K⁻¹) and has part of the free activation energy (ΔG^‡ = 75 kJ mol⁻¹; 298 K) shifted from entropy to enthalpy (ΔΔH^‡ = 16 kJ mol⁻¹). Substrate isotope effects are temperature independent for wild-type UGAepi in both solvents yet decrease for the positioning-impaired R88A variant in ²H₂O. This evidence suggests that ²H₂O dampens protein fluctuations, thereby leading to enthalpy-entropy compensation in both enzymes and to conformational sampling in the R88A variant.