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 (D2O)k(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 kcat or k(cat)/K-M value significantly, establishing that nitroethane binding is at equilibrium and that product release does not limit k(cat). (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.