Solvent isotope and viscosity effects on the steady-state kinetics of the flavoprotein nitroalkane oxidase

The flavoprotein nitroalkane oxidase catalyzes the oxidative denitrification of a broad range of primary and secondary nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. With nitroethane as substrate the ^D2O(k_cat/K_M) value is 0.6 and the ^D2Ok_cat value is 2.4. The k_cat proton inventory is consistent with a single exchangeable proton in flight, while the k_cat/K_M is consistent with either a single proton in flight in the transition state or a medium effect. Increasing the solvent viscosity did not affect the k_cat or k_cat/K_M value significantly, establishing that nitroethane binding is at equilibrium and that product release does not limit k_cat.