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 (\({\Delta {{{{{\rm{H}}}}}}}^{{{\ddagger}} }\) = 54 kJ mol⁻¹), significant loss in entropy (\(-T{\Delta {{{{{\rm{S}}}}}}}^{{{\ddagger}} }\) = 20 kJ mol⁻¹; 298 K) and negative activation heat capacity (\({\Delta {{{{{\rm{C}}}}}}}_{{{{{{\rm{p}}}}}}}^{{{\ddagger}} }\) = −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.