Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The FAD-containing NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the four-electron reduction of O2----2H2O, has been purified by an improved procedure for analyses of its structural and redox properties. The enzyme is apparently a dimer of two identical subunits, each containing 1 mol of FAD. Dithionite reduction of the enzyme proceeds in two distinct phases corresponding to approximately 0.5 and 1.1 eq/FAD, respectively. Thiol assays of the NADH oxidase, reduced anaerobically with 1 eq of NADH/FAD prior to denaturation, are consistent with the presence of a single redox-active cysteinyl residue/subunit. Analysis of the cysteinyl peptides of the oxidase, identified in tryptic digests of the enzyme labeled metabolically with [35S]cysteine, reveals a sequence which is closely related to the redox-active cysteinyl peptide sequence recently determined for the streptococcal flavoprotein NADH peroxidase. A second cysteinyl peptide sequence, when aligned with residues 3-17 of the peroxidase NH2-terminal sequence, reveals identity in 7 of 15 positions and satisfies several of the criteria described for ADP-binding structures. Additional probes of the structural and redox properties of the NADH oxidase, including visible circular dichroism spectroscopy and sensitivity to inactivation by hydrogen peroxide, provide further evidence for a fundamental structural connection between flavin-dependent NADH oxidase and peroxidase functions.
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PMID:The streptococcal flavoprotein NADH oxidase. I. Evidence linking NADH oxidase and NADH peroxidase cysteinyl redox centers. 251 Nov 95

Previous results from this laboratory demonstrated that glutathione concentrations decrease in aging mouse tissues. In this investigation glutathione (GSH) peroxidase and glutathione (GSSG) reductase activities were measured in tissues of standardized aging mice. Methods were validated for the quantitative determination of both enzymes in liver, kidney and heart tissues. GSH peroxidase activities were 27-53% lower in liver, kidney and heart of very old (36 months) mice compared to mature (10 months) mice (P less than 0.01). The same aging decreases were found with either hydrogen peroxide or cumene hydroperoxide as substrate. In a similar way GSSG reductase activities in liver and kidney were 25-28% lower in the old versus mature mice (P less than 0.01), but heart levels were unchanged. Further the lower GSSG reductase levels were unaffected by FAD supplementation in vitro. The changes in specific activity for both enzymes were not due to changes in organ weights and total protein contents, which were constant from 10 to 36 months of age. These decreases in GSH peroxidase and GSSG reductase do not account for the lower GSH levels in aging. Of special importance, however, is that these decreases indicate that detoxification via glutathione peroxidase and glutathione reductase could be impaired in senescence.
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PMID:Glutathione peroxidase and reductase activities in the aging mouse. 398 84

Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The K(m) and V(max.) values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide.
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PMID:The isolation and properties of phenylalanine hydroxylase from rat liver. 485 20

Very small quantities of FAD were able to reactivate apo-D-amino acid oxidase. In the presence of D-alanine, luminol, horseradish peroxidase, and an excess of the apoenzyme, a quantitative luminometric determination of FAD was possible. The maximal photon emission measured in a bicarbonate buffer, pH 9.2, at 37 degrees C was proportional to the amount of FAD added. FMN, riboflavin, or 5-deazaflavin produced no chemiluminescence and had no inhibitory effect in the assay when added together with FAD. With this method, FAD could be quantitatively determined with high accuracy in perchloric acid extracts of animal tissue and bacteria.
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PMID:Luminometric determination of FAD in subpicomole quantities. 613 73

An L-amino acid oxidase (L-amino-acid oxygen oxidoreductase (deaminating), EC 1.4.3.2) from the blue-green alga Anacystis nidulans has been purified to homogeneity with an overall yield of about 10%. Purification included ammonium sulfate fractionation and CM-Sephadex, DEAE-Sephadex, and hydroxyapatite chromatography. The purified enzyme has an absorption spectrum which is characteristic of a flavoprotein, and contains 1 mol FAD per mol enzyme. The native enzyme has a molecular weight of 98 000 as determined by gel exclusion chromatography. Electrophoresis in SDS-polyacrylamide gels gives a single protein band corresponding to a molecular weight of 49 000, which suggests that the native enzyme is composed of 2 subunits of equal molecular weight. As previously demonstrated, the enzyme catalyzes the oxidative deamination of the basic amino acids: L-arginine, L-lysine, L-ornithine and L-histidine. In the presence of catalase and of any of these amino acids, 0.5 mol O2 is consumed, and 1 mol ammonia is formed for each mol amino acid oxidized. HCN is formed from L-histidine when the L-amino acid oxidase is supplemented with peroxidase. In addition to the unusual substrate specificity of this L-amino acid ozidase, it also has an unusual set of inhibitors including o-phenanthroline as well as divalent cations of which Cu2+, Zn2+, and Cd2+ are the most effective ones, but Mg2+ and Ca2+ also inhibit. This inhibition can be reversed by chelating agents such as EDTA. ATP and ADP, but not AMP, can also overcome the inhibition caused by Mg2+, for example. The inhibitory effect of cations can be demonstrated in vivo.
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PMID:Some properties of a basic L-amino-acid oxidase from Anacystis nidulans. 676 43

The Prostatic-Group-Label Immunoassay (PGLIA) technique has been incorporated into a reagent-strip format. We report use of flavin N6-(N'-2,4-dinitrophenyl-6-aminohexyl)adenine dinucleotide (DNP-FAD) as the prosthetic group derivative and 6-N-(2,4-dinitrophenyl)aminohexanoic acid (DNP-caproate) as the competing ligand. DNP-FAD not bound by antibody combines with glucose oxidase apoenzyme, which then reacts with glucose and oxygen, and gives color through a peroxidase-linked system. The rate of color generation is thus a function of the DNP-caproate concentration. PGLIA reagent strips are prepared by sequential impregnations of filter paper with an acetone solution of indicator (3,3',5,5'-tetramethylbenzidine); an aqueous solution containing glucose oxidase apoenzyme, the rest of the color generation system, stabilizers, and antibody to DNP; and a solution of DNP-FAD in n-propanol. This preparation permits effective antibody binding, and prevents premature interaction of immunoassay components. A quantitative color response to concentrations of DNP-caproate in the range of 1 to 8 mumol/L was demonstrated with these reagent strips. Prototype PGLIA reagent strips for theophylline and phenytoin have been successfully developed by substituting the appropriate FAD derivative and antibody for the corresponding reagents in the DNP model system.
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PMID:Adaptation of Prostatic-Group-Label Homogeneous Immunoassay to reagent-strip format. 726 29

Peroxidase (E.C. 1.11.1.7., hydrogen donor oxidoreductase) is widely distributed and has been isolated from many higher plants (1). The wide distribution of the enzyme suggests that it could be of great biological importance. However the role that it plays in metabolism is not clear due to the large number of reactions it catalyzes and the considerable number of isozymic species (2). In tomato plants, Evans and Aldridge (3) separated out six isoperoxidases and in a later paper Evans reported 12 isoperoxidases from tomato shoots (4). A homogeneous tomato fruit peroxidase isozyme was obtained by Jen et al. (5) using hydrophobic chromatography. Isozymes were not detected in Euphorbia characias peroxidase (6), in Ipomoea batatas peroxidase (7) and in Hordeum vulgare peroxidase (8). The simultaneous presence of Cu (II) amine oxidase and peroxidase in cell walls suggests that the peroxide generated on oxidation of the amines could be utilized by the peroxidase (6,8,9). In the graminea Oryza sativa, widely distributed, an FAD amine oxidase is present that oxidizes diamines (10). In this plant we also found two isoperoxidases called perox I and II. Only perox I was purified to homogeneity and its enzymatic, physical and chemical properties have been studied.
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PMID:Fractionation and characterization of two forms of peroxidase from Oryza sativa. 760 69

The flavoprotein NADH peroxidase from Enterococcus faecalis 10C1 has been shown to contain, in addition to FAD, an unusual cysteine-sulfenic acid (Cys-SOH) redox center. The non-flavin center cycles between reduced (Cys-SH) and oxidized (Cys-SOH) states, and the 2.16 A crystal structure of the non-native cysteine-sulfonic acid (Cys-SO3H) form of the wild-type peroxidase supports the proposed catalytic role of Cys42. In this study, we have employed a site-directed mutagenesis approach in which Cys42 is replaced with Ser and Ala, neither side chain of which is capable of redox activity. Reductive titrations of both C42S and C42A mutants lead directly to full FAD reduction with 1 equiv of either dithionite or NADH, consistent with elimination of the Cys-SOH center. Direct determinations of the redox potentials for the FAD/FADH2 couples yield values of -219 and -197 mV, respectively, for C42S and C42A peroxidases, indicating that the presence of Cys42-SH in the two-electron-reduced wild-type enzyme lowers the flavin potential by approximately 100 mV. Anaerobic stopped-flow analyses of the reduction of C42S and C42A peroxidases by NADH demonstrate that in both cases flavin reduction is rapid; these results are confirmed by enzyme-monitored, steady-state kinetic analyses which, in addition, give turnover numbers approximately 0.04% that of wild-type enzyme. These results are entirely consistent with the role proposed for Cys42 in the catalytic redox cycle of wild-type NADH peroxidase and indirectly support its function as a peroxidatic center in the homologous NADH oxidase.
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PMID:Analysis of the kinetic and redox properties of NADH peroxidase C42S and C42A mutants lacking the cysteine-sulfenic acid redox center. 781 35

The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the peroxidase-catalysed biosynthesis of thyroid hormones. The electron transfer from NADPH to O2 catalysed by this system was studied by using diphenyleneiodonium (DPI), an inhibitor of flavo- and haemo-proteins. The prosthetic group of the H2O2 generator was removed by incubation with 5 mM CHAPS at 40 degrees C, and an active holoenzyme was reconstituted with FAD, but not with FMN. The H2O2-generating system also had an intrinsic Ca(2+)-dependent NADPH:ferricyanide reductase activity which is probably linked to its flavodehydrogenase component (or domain). Both activities, H2O2 production and ferricyanide reductase activity, were inhibited by DPI, with similar K1/2 (2.5 nmol/mg of protein). DPI only inhibited a system reduced with NADPH in the presence of Ca2+. NADPH could not be replaced by NADP+, NADH or sodium dithionite, suggesting the need for specific mild reduction of a redox centre in a particular conformation. Ferricyanide protected both activities against inhibition by DPI; the NADPH:ferricyanide reductase activity was completely protected at a ferricyanide concentration 20 times lower than that needed to protect the H2O2 formation, implying at least two target sites for DPI. One might be the flavodehydrogenase component; the other was beyond, on the entity which transfers the electrons to O2. This second site has not been identified.
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PMID:The Ca2+/NADPH-dependent H2O2 generator in thyroid plasma membrane: inhibition by diphenyleneiodonium. 803 94

Heterodisulfide reductase catalyzes the terminal step in the energy-conserving electron-transport chain in methanogenic Archaea. The heterodisulfide reductase activity of the membrane fraction of methanol-grown Methanosarcina barkeri was solubilized by Chaps. Chromatography on Q-Sepharose and Superdex-200 yielded a high-molecular-mass fraction (> 700 kDa) which was dissociated by dodecyl beta-D-maltoside. After chromatography on Q-Sepharose, an active heterodisulfide reductase preparation was obtained which was composed of only two different subunits of apparent molecular masses 46 kDa and 23 kDa. For each 69 kDa, the enzyme contained 0.6 mol cytochrome b, 0.2 mol FAD, 20 mol non-heme iron and 20 mol acid-labile sulfur. The 23-kDa subunit possessed heme-derived peroxidase activity, showing that this polypeptide is the cytochrome b. The purified enzyme contained the cytochrome b in the reduced form. Upon addition of the heterodisulfide of coenzyme M and N-7-mercaptoheptanoylthreonine phosphate the cytochrome was instantaneously oxidized, indicating that the cytochrome b served as electron donor for heterodisulfide reduction.
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PMID:Purification of a two-subunit cytochrome-b-containing heterodisulfide reductase from methanol-grown Methanosarcina barkeri. 817 66


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