flavin-adenine-dinucleotide and 3-aminopyridine-adenine-dinucleotide

NADH peroxidase is a flavoenzyme having a single redox-active thiol, Cys42, that cycles between sulfenate and thiol forms in the NADH-dependent reduction of hydrogen peroxide. NADH peroxidase catalyzes the NADH-dependent reduction of quinones with turnover numbers between 1.2 and 3.9 s-1, per mole of FAD, at pH 7.5. The bimolecular rate constants for quinone reduction, V/K, ranged from 4.3 x 10(3) to 6.0 x 10(5) M-1 s-1 for 14 quinones whose redox potentials varied between -0.41 and 0.09 V. The logarithms of the V/K values for these quinones are hyperbolically dependent on their single-electron reduction potentials (E7(1). One-electron reduction of benzoquinone accounts for about 50% of the total electron transfer catalyzed by NADH peroxidase at pH 7, with the remainder of the reduction being catalyzed by a two-electron (hydride) transfer. Cys42 can be irreversibly oxidized to the sulfonate by hydrogen peroxide, with inactivation of the peroxidatic activity of the enzyme. The residual quinone reductase activity of NADH peroxidase which has undergone oxidative inactivation of the active site Cys42 indicates that this residue is not involved in the reduction of the quinones. Product inhibition studies suggest the possibility of overlap of the pyridine nucleotide and quinone binding sites in the reduced enzyme at low pH values. The pH dependence of the maximum velocity of naphthoquinone reduction shows that deprotonation of an enzymic group, exhibiting a pK value of ca. 6.2, decreases the maximal velocity. Primary deuterium kinetic isotope effects on V and V/K for quinone-dependent NADH oxidation increase upon protonation of a group, exhibiting a pK value of 6.4.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Binding Sites; Catalysis; Deuterium; Electron Transport; Enterococcus faecalis; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Kinetics; NAD; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Oxidation-Reduction; Peroxidases; Quinones

Milk xanthine oxidase (XO) has been prepared in a dehydrogenase form (XDH) by purifying the enzyme in the presence of 2.5 mM dithiothreitol. Unlike XO, which reacts rapidly only with oxygen and not with NAD, the XDH form of the enzyme reacts rapidly with NAD. XDH has a turnover number for the NAD-dependent conversion of xanthine to urate of 380 mol/min/mol at pH 7.5, 25 degrees C, with a Km = < or = 1 microM for xanthine and a Km = 7 microM for NAD, but has very little O2-dependent activity. There is evidence that the two forms of the enzyme have different flavin environments: XDH stabilizes the neutral form of the flavin semiquinone and XO does not. Further, XDH binds the artificial flavin 8-mercapto-FAD in its neutral form, shifting the pK of this flavin by 5 pH units, while XO binds 8-mercapto-FAD in its benzoquinoid anionic form. XDH can be converted back to the XO form by the addition of three to four equivalents of the disulfide-forming reagent 4,4'-dithiodipyridine, suggesting that, in the XDH form of the enzyme, disulfide bonds are broken; this may cause a conformational change which creates a binding site for NAD and changes the protein structure near the flavin.

Topics: Animals; Cattle; Disulfides; Flavin-Adenine Dinucleotide; Glutathione; Kinetics; Milk; NAD; Oxidation-Reduction; Photochemistry; Pyridines; Superoxides; Xanthine Dehydrogenase

Chicken liver xanthine dehydrogenase can be partially reduced by either xanthine or NADH. Reduction to approximately the 2-electron-reduced level occurs with NADH, and reduction beyond the 2-electron level occurs with xanthine. In both cases, the reaction is triphasic. The first and third phases are dependent on reductant concentration, whereas the second phase is not. Oxidation of fully (6-electron) reduced xanthine dehydrogenase by either urate or NAD is monophasic and dependent on the oxidant concentration. Oxidation stops at about the same level of reduction that was reached by the corresponding reductant. The position of this end point is sensitive to the potential of the reactants but is relatively insensitive to excess concentrations of oxidant or reductant. NADH binding to 2-electron-reduced enzyme is implicated in fixing the end point position in those reactions involving pyridine nucleotides, whereas urate binding is involved in fixing the end point of those reactions involving xanthine and urate.

Topics: Animals; Chickens; Dithionite; Electron Transport; Flavin-Adenine Dinucleotide; Ketone Oxidoreductases; Kinetics; Liver; NAD; Oxidation-Reduction; Spectrophotometry; Uric Acid; Xanthine; Xanthine Dehydrogenase; Xanthines

Topics: Alkylation; Animals; Dihydrolipoamide Dehydrogenase; Dose-Response Relationship, Drug; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Macromolecular Substances; Myocardium; NAD; Spectrum Analysis; Swine