Abstract：
:The flavoprotein nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to aldehydes and ketones, respectively, transferring electrons to oxygen to form hydrogen peroxide. The steady-state kinetic mechanism of the active flavin 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 the 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, respectively. The free reduced enzyme forms a dead-end complex with nitroethane, with a K(ai) value of 30 +/- 6 mM. Acetaldehyde and butyraldehyde are noncompetitive inhibitors versus nitroethane due to formation of a dead-end complex between the oxidized enzyme and the product. Acetaldehyde is an uncompetitive inhibitor versus oxygen, indicating that an irreversible isomerization of the free reduced enzyme occurs before the reaction with oxygen. Addition of unprotonated imidazole results in a 5-fold increase in the V(max) value, 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. These results are consistent with the isomerization of the free reduced enzyme being about 80% rate-limiting for catalysis and with a model in which unprotonated imidazole accelerates the rate of isomerization.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Gadda G,Fitzpatrick PFdoi
10.1021/bi9922547subject
Has Abstractpub_date
2000-02-15 00:00:00pages
1400-5issue
6eissn
0006-2960issn
1520-4995pii
bi9922547journal_volume
39pub_type
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