An NADH oxidase was purified to homogeneity from Leuconostoc mesenteroides with a specific activity 100-fold higher than that of the crude extract.
1. The purified NADH oxidase was an acidic protein having an s⁰₂₀, w of 5.49 S and a molecular weight of 104, 000, consisting of a dimer with 53, 000 subunit size.
2. The enzyme could use O₂, dichlorophenolindophenol and methylene blue as oxidants, but not H₂O₂, cytochrome c, or ferricyanide. The physiological substrate was β-NADH (K_m=0.12mM) with O₂ as the oxidant, probably forming H₂O, rather than H₂O₂. Activity toward α-NADH was observed (K_m=0.14mM), but the maximum velocity was 3 orders of magnitude lower than that with β-NADH. α-NADPH and β-NADPH were inert for the reaction.
3. The enzyme showed a flavoprotein absorption spectrum with maxima at 273, 379, and 450 nm with a shoulder at 465 nm: the absorption at 450-465 nm disappeared on adding excess NADH or hydrosulfite. One mol of the holoenzyme contained approximately 2 mol of FAD. The apoenzyme was obtained by treatment with EDTA-KBr solution and could be reconstituted partially by adding FAD, but not riboflavin or FMN.
4. The maximum activity of the reaction was observed at pH 6.5 in a temperature range of 35-45°C. The activation energy was estimated to be 3.77 kcal/mol.
5. The enzyme was inhibited by SH reagents, quinacrine, quinine, and Cue⁺, but not by EDTA. Adenine and its nucleoside 5'-di- and triphosphates showed competitive inhibitions, while various metabolites, such as H₂O₂, FDP, acetyl phosphate, lactate, ethanol, and acetate, did not affect the reaction.