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

Abstract

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 ( <math xmlns="http://www.w3.org/1998/Math/MathML"> <msup><mrow><mi>Δ</mi> <mi>H</mi></mrow> <mrow><mo>‡</mo></mrow> </msup> </math>  = 54 kJ mol^-1), significant loss in entropy ( <math xmlns="http://www.w3.org/1998/Math/MathML"><mo>-</mo> <mi>T</mi> <msup><mrow><mi>Δ</mi> <mi>S</mi></mrow> <mrow><mo>‡</mo></mrow> </msup> </math>  = 20 kJ mol^-1; 298 K) and negative activation heat capacity ( <math xmlns="http://www.w3.org/1998/Math/MathML"> <msubsup><mrow><mi>Δ</mi> <mi>C</mi></mrow> <mrow><mi>p</mi></mrow> <mrow><mo>‡</mo></mrow> </msubsup> </math>  = -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.