Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis

Understanding enzyme catalysis as connected to protein motions is a major challenge. Here, based on temperature kinetic studies combined with isotope effect measurements, we obtain energetic description of C-H activation in NAD-dependent UDP-glucuronic acid C4 epimerase. Approach from the ensemble-averaged ground state (GS) to the transition state-like reactive conformation (TSRC) involves, alongside uptake of heat ( Δ H ‡ = 54 kJ mol −1 ), significant loss in entropy ( − T Δ S ‡ = 20 kJ mol −1 ; 298 K) and negative activation heat capacity ( Δ C p ‡ = −0.64 kJ mol −1 K −1 ). Thermodynamic changes suggest the requirement for restricting configurational freedom at the GS to populate the TSRC. Enzyme variants affecting the electrostatic GS preorganization reveal active-site interactions important for precise TSRC sampling and H-transfer. Collectively, our study captures thermodynamic effects associated with TSRC sampling and establishes rigid positioning for C-H activation in an enzyme active site that requires conformational flexibility in fulfillment of its natural epimerase function. Enzymes involve structural flexibility in their function, but understanding enzyme catalysis as connected to protein motions is a major challenge. Here, the authors obtain energetic description of C-H activation in nicotinamide coenzyme-dependent UDP-glucuronic acid C4 epimerase based on temperature kinetic studies and isotope effect measurements.