Purification and characterization of NADH oxidase from a strain of Leuconostoc mesenteroides.
An NADH oxidase was purified to homogeneity from Leuconostoc mesenteroides with a specific activity 100-fold higher than that of the crude extract. The purified NADH oxidase was an acidic protein having an S0 20,W of 5.49S and a molecular weight of 104,000, consisting of a dimer with 53,000 subunit size. The enzyme could use O2, dichlorophenolindophenol and methylene blue as oxidants, but not H2O2, cytochrome c, or ferricyanide. The physiological substrate was beta-NADH (Km = 0.12 mM) with O2 as the oxidant, probably forming H2O, rather than H2O2. Activity toward alpha-NADH was observed (Km = 0.14 mM), but the maximum velocity was 3 orders of magnitude lower than that with beta-NADH. alpha-NADPH and beta-NADPH were inert for the reaction. 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. The maximum activity of the reaction was observed at pH 6.5 in a temperature range of 35-45 degrees C. The activation energy was estimated to be 3.77 kcal/mol. The enzyme was inhibited by SH reagents, quinacrine, quinine, and Cu2+, 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.
D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D014461:Ultracentrifugation
D009097:Multienzyme Complexes; D012265:Ribonucleotides; D013439:Sulfhydryl Reagents; D009243:NAD; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases