Iso-mechanism of nitroalkane oxidase: 1. Inhibition studies and activationby imidazole

The flavoprotein nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to aldehydes and ketones, respectively, transferrin g electrons to oxygen to form hydrogen peroxide. The steady-stare kinetic m echanism of the active Ravin adenine dinucleotide-(FAD-) containing form of the enzyme has been determined with nitroethane at pH 7 to be bi-ter ping- pong, with oxygen reacting with the free reduced enzyme after release of th e aldehyde product. The V-max value is 5.5 +/- 0.3 s(-1) and the K-m values for nitroethane and oxygen are 3.3 +/- 0.6 and 0.023 +/- 0.007 mM, respect ively. The free reduced enzyme forms a dead-end complex with nitroethane, w ith a K-ai value of 30 +/- 6 mM. Acetaldehyde and butyraldehyde are noncomp etitive inhibitors versus nitroethane due to formation of a dead-end comple x between the oxidized enzyme and the product. Acetaldehyde is an uncompeti tive inhibitor versus oxygen, indicating that an irreversible isomerization of the free reduced enzyme occurs before the reaction with oxygen. Additio n of unprotonated imidazole results in a fi-fold increase in the V-max valu e, while the V/K values for nitroethane and oxygen are unaffected. A 5-fold increase in the K-ai value for nitroethane and a 6.5-fold increase in the K-ii value for butyraldehyde are observed in the presence of imidazole. The se results are consistent with the isomerization of the free reduced enzyme being about 80% rate-limiting for catalysis and with a model in which unpr otonated imidazole accelerates the rate of isomerization.