Thermodynamic analysis of remote substrate binding energy in 3α-hydroxysteroid dehydrogenase/carbonyl reductase catalysis

Abstract The binding energy of enzyme and substrate is used to lower the activation energy for the catalytic reaction. 3α-HSD/CR uses remote binding interactions to accelerate the reaction of androsterone with NAD+. Here, we examine the enthalpic and entropic components of the remote binding energy in the 3α-HSD/CR-catalyzed reaction of NAD+ with androsterone versus the substrate analogs, 2-decalol and cyclohexanol, by analyzing the temperature-dependent kinetic parameters through steady-state kinetics. The effects of temperature on kcat/Km for 3α-HSD/CR acting on androsterone, 2-decalol, and cyclohexanol show the reactions are entropically favorable but enthalpically unfavorable. Thermodynamic analysis from the temperature-dependent values of Km and kcat shows the binding of the E-NAD+ complex with either 2-decalol or cyclohexanol to form the ternary complex is endothermic and entropy-driven, and the subsequent conversion to the transition state is both enthalpically and entropically unfavorable. Hence, solvation entropy may play an important role in the binding process through both the desolvation of the solute molecules and the release of bound water molecules from the active site into bulk solvent. As compared to the thermodynamic parameters of 3α-HSD/CR acting on cyclohexanol, the hydrophobic interaction of the B-ring of steroids with the active site of 3α-HSD/CR contributes to catalysis by increasing exclusively the entropy of activation (ΔTΔS‡ = 1.8 kcal/mol), while the BCD-ring of androsterone significantly lowers ΔΔH‡ by 10.4 kcal/mol with a slight entropic penalty of −1.9 kcal/mol. Therefore, the remote non-reacting sites of androsterone may induce a conformational change of the substrate binding loop with an entropic cost for better interaction with the transition state to decrease the enthalpy of activation, significantly increasing catalytic efficiency.