Comparison of enzymatic and non-enzymatic nitroethane anion formation: Thermodynamics and contribution of tunneling

In the reaction of nitroalkane oxidase (NAO), the oxidation of nitroalkanes to the corresponding aldehydes or ketones is initiated by the deprotonation of the neutral nitroalkane. The energetics of nitroethane ionization for both the enzymatic and non-enzymatic reactions have been determined by measuring rate constants as a function of temperature. At 25 °C, the rate constant for the acetate-catalyzed reaction is a billionfold smaller than the k_cat/K_m value for NAO. This corresponds to a difference of 12.3 kcal/mol in the free energy of activation that is largely due to a difference in the activation enthalpy. Analysis of the temperature dependence of the deuterium kinetic isotope effects on the reactions yields similar ΔE_a and A_H/A_D values for the acetate, phosphate, and NAO-catalyzed reactions that fall within the semiclassical limits, consistent with similar contributions of tunneling to the enzymatic and non-enzymatic reactions.