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)

Bovine serum amine oxidase is inhibited by benzylhydrazine (BHy), but recovers full activity after a few hours incubation [Hucko-Haas & Reed (1970) Biochem. Biophys. Res. Commun. 38, 396-400]. The first phase of the process, requiring about 15 min, was found to consist of a mechanism-based hydrazine-transfer reaction leading to formation of the hydrazine-bound enzyme, benzaldehyde and H2O2. At variance with the enzymic process, the reaction with O2 preceded the benzaldehyde release. Two reaction intermediates could be characterized by optical spectroscopy and were assigned as the azo derivative and the benzaldehyde hydrazone, the latter one probably being involved in the reaction with O2. No reduction of Cu was detected at any stage. The hydrazine adduct could also be obtained by stoichiometric reaction of hydrazine with the native enzyme. The decay of this species occurred in about 8 h and was not studied in detail. The Cu-binding inhibitor NN-diethyldithiocarbamate affected the BHy reaction by stabilizing the benzaldehyde hydrazone form as against the azo derivative and the reaction with O2. However, under these same conditions the initial spectroscopic properties of the diethyldithiocarbamate adduct were recovered if the oxidase was left overnight. The reaction with O2 was abolished only upon removal of at least one Cu atom from the enzyme. On the basis of the failure to detect any change of Cu redox state and the enzyme behaviour in the presence of inhibitors, a reaction mechanism involving the formation of a hydroperoxy intermediate, as in the FAD-containing enzymes, is tentatively proposed.
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PMID:Benzylhydrazine as a pseudo-substrate of bovine serum amine oxidase. 254 50

A new flavoenzyme using molecular oxygen to oxidize L-glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2-oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including D-glutamic acid, L-glutamine and L-aspartic acid, only L-glutamic acid is converted. The molecular mass of the enzyme was estimated to be about 90 kDa by gel chromatography and 50 kDa by SDS/PAGE. The subunit contains 1 molecule noncovalently bound FAD. The absorption spectrum shows maxima at 273, 355 and 457 nm and the isoelectric point is at pH 6.2. The Km value for L-glutamic acid in air-saturated phosphate pH 7.0 was estimated to be 1.1 mM, the Km for oxygen was calculated to be 1.86 mM at saturating concentration of L-glutamic acid. The enzymic reaction is inhibited by Ag+ and Hg2+ ions. The enzyme described here distinctly differs from two microbial L-glutamate oxidases purified hitherto, with regard to extremely high substrate specificity and to the subunit structure.
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PMID:A novel L-glutamate oxidase from Streptomyces endus. Purification and properties. 273 5

6-Thiocyanatoflavins have been found to be susceptible to nucleophilic displacement reactions with sulfite and thiols, yielding respectively the 6-S-SO3--flavin and 6-mercaptoflavin, with rate constants at pH 7.0, 20 degrees C, of 55 M-1 min-1 for sulfite and 1000 M-1 min-1 for dithiothreitol. The 6-SCN-flavin binds tightly to riboflavin-binding protein as the riboflavin derivative, to apoflavodoxin, apo-lactate oxidase, and apo-Old Yellow Enzyme as the FMN derivative, and to apo-D-amino acid oxidase as the FAD derivative. The riboflavin-binding protein derivative is inaccessible to dithiothreitol attack, and the lactate oxidase and D-amino acid oxidase derivatives show only limited accessibility. However, the flavodoxin and Old Yellow Enzyme derivatives react readily with dithiothreitol, indicating that the flavin 6-position is exposed to solvent in these proteins. The lactate oxidase and D-amino acid oxidase derivatives convert slowly but spontaneously to the 6-mercaptoflavin enzyme forms in the absence of any added thiol, indicating the presence of a thiol residue in the flavin binding site of these proteins. The reaction rates have been investigated of 6-mercaptoflavins with iodoacetamide, N-ethylmaleimide, methyl methanethiosulfonate, H2O2, and m-chloroperbenzoate, in both the free and protein-bound state. The results confirm the conclusions drawn from the studies with 6-SCN-flavins described above and from 6-N3-flavins [Massey, V., Ghisla, S., & Yagi, K. (1986) Biochemistry (preceding paper in this issue)]. The spectral properties of the protein-bound 6-mercaptoflavin vary widely among the five proteins studied and show stabilization of the neutral flavin with flavodoxin and riboflavin-binding protein and of the anionic species by Old Yellow Enzyme, lactate oxidase, and D-amino acid oxidase. In the case of the latter two enzymes, the stabilization appears to be due to interaction of the negatively charged flavin with a positively charged protein residue located near the flavin pyrimidine ring. This positively charged residue appears to be responsible also for the strong stabilization of the two-electron oxidation state of the mercaptoflavin as the 6-S-oxide. With the other flavoproteins studied this oxidation level is stabilized as the 6-sulfenic acid or 6-sulfenate.
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PMID:6-Thiocyanatoflavins and 6-mercaptoflavins as active-site probes of flavoproteins. 287 64

An H2O-forming NADH oxidase from Streptococcus faecalis, recently described [Hoskins, D. D., Whiteley, H. R. and Mackler, B. (1962) J. Biol. Chem. 237, 2647-2651], has been isolated as a uniform protein with specific activity 690 U/mg in a total yield of 50% by a two-step affinity chromatography procedure. The enzyme is metal-free and has a molecular mass of about 51 000 Da and probably consists of a single polypeptide chain. As shown by fluorimetric titration, the prosthetic group is 1 mol FAD/mol protein. The affinity behaviour of the enzyme gives evidence for the existence of a dinucleotide-binding domain capable of binding NADH or FAD. The enzyme is specific for NADH (Km = 4.1 X 10(-5) M), NADPH is not oxidized. O2 is the preferred electron acceptor, in addition FAD and, very slowly, one-electron acceptors are reduced. It is not clear whether the reduction of FAD proceeds through the dinucleotide-binding site or by exchange of the prosthetic group. The stoichiometry of the reaction with O2 corresponds to the consumption of 2 mol NADH/mol O2, and only H2O is formed (2 NADH + 2 H+ + O2----2 NAD+ + 2 H2O). Neither H2O2 nor O2.- is detected as intermediate and H2O2 cannot replace O2 as an oxidant. The enzyme can, mainly in its reduced state, be inhibited by -SH reagents. Spectral data give no evidence for the existence of radical intermediates during reduction. The enzyme can obviously accept more than two electrons/mol. On the basis of these data two possible reaction mechanisms are discussed. A proposal for the biological purpose of the reaction is made.
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PMID:Isolation and properties of an H2O-forming NADH oxidase from Streptococcus faecalis. 308 30

The flavin-containing NADH peroxidase of Streptococcus faecalis 10C1, which catalyzes the reaction: NADH + H+ + H2O2----NAD+ + 2H2O, has been purified to homogeneity in our laboratory for analyses of both its structure and redox behavior. Our findings indicate that the enzyme is a tetramer of four apparently identical subunits (Mr = 46,000/subunit), each containing one FAD coenzyme and a second non-flavin, nonmetal redox center. There is no evidence of nonequivalence among the flavins. Dithionite reduction of the enzyme occurs in two steps, with end points of 0.96 and 2.05 eq/FAD. The first step generates a two-electron reduced form of the enzyme (EH2) which is spectrally identical with that generated by aerobic addition of NADH. Our studies suggest that the long-wavelength absorbance band (lambda max approximately 540 nm) exhibited by this form results from charge-transfer interaction between the reduced non-flavin redox center and the oxidized flavin. A second type of long-wavelength charge-transfer absorbance band (lambda max approximately 770 nm) is generated on anaerobic addition of 1 eq of NADH to EH2 and results from interaction between oxidized FAD and the reduced pyridine nucleotide. Either the EH2 X NAD+ or the EH2 X NAD+ X NADH forms may be involved in the catalytic mechanism of the enzyme, as both are reactive with hydrogen peroxide.
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PMID:Interactions of pyridine nucleotides with redox forms of the flavin-containing NADH peroxidase from Streptococcus faecalis. 309 21

A new FAD-dependent monooxygenase, 4-aminobenzoate hydroxylase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate and forms 4-hydroxyaniline in the presence of NAD(P)H and O2 has been purified to homogeneity by ammonium sulfate fractionation, affinity chromatography, chromatofocusing, and Sephadex G-100 chromatography from Agaricus bisporus, a common edible mushroom. The molecular weight of the enzyme, which consists of a single polypeptide, is 49,000. The enzyme contains 0.91 mol of FAD/mol of enzyme. Stoichiometric studies show that 1 mol of 4-aminobenzoate is converted to an equimolecular amount of 4-hydroxyaniline and CO2 with the consumption of 1 mol each of NADH and molecular oxygen. Results obtained isotopically with 18O2 show that one atom of molecular oxygen is incorporated into 4-hydroxyaniline formed from 4-aminobenzoate. The enzyme is most active between pH 6.5 and 8.0 in the oxidation of NADH and between pH 6.0 and 7.5 in the case of NADPH. The Km values for 4-aminobenzoate, NADH, and O2 are 20.4, 13.6, and 200 microM, respectively, and that for NADPH is 133 microM. Other substituted benzoates with free amino and carboxyl groups in the ortho or para position (e.g. 4-aminosalicylate and anthranilate) serve as substrates for hydroxylation, but, in these cases, H2O2 is formed simultaneously with the hydroxylation. The enzyme is insensitive to the chelators of iron and copper, sodium arsenite, and KCN. Heavy metal ions and p-chloromercuribenzoate severely inhibit the enzyme enzyme
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PMID:Purification and properties of 4-aminobenzoate hydroxylase, a new monooxygenase from Agaricus bisporus. 348 13

Pig blood neutrophils were briefly activated by various fatty acids and then fractionated into membrane vesicles with different NADPH oxidase activities. Treatment of these membranes with a detergent, octyl glucoside, resulted in a high yield of solubilized oxidase, which was subjected to isoelectric focusing on gels (pI 4.0-8.0). 1) A distinct band staining with NADPH-nitroblue tetrazolium focused at pI 5.0. The enzyme (pI 5.0) showed high specificity for NADPH and similar characteristics to the oxidase involved in the respiratory burst. 2) The enzyme was extracted from gel slices and analyzed. When measured promptly after its extraction, its NADPH oxidase activity was high, but there was apparent superoxide dismutase-insensitive cytochrome c reduction, probably due to direct electron transfer to the heme protein. However, it could produce superoxide anion (O2-) under some micelle conditions. 3) Therefore, the formation of the enzyme-substrate complex of yeast cytochrome c peroxidase was employed for the detection of H2O2. A fresh extract of stimulated cells catalyzed equimolar NADPH oxidation and H2O2 production of 306 and 300 nmol min-1 (mg protein)-1, respectively. The Km value of the enzyme for NADPH was 30 +/- 13 (S.D.) microM. The recovery of the extract (pI 5.0) was 19% of the total activity. 4) The enzyme extract contained 1.1-1.9 nmol of FAD/mg of protein, giving a turnover number of 300-600 min-1 in terms of O2- generation/FAD. No heme protein was found in the enzyme. The enzyme was mainly of 67-kDa molecular mass.
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PMID:The respiratory burst oxidase of neutrophils. Separation of an FAD enzyme and its characterization. 362 61

5-Pyridoxic-acid oxygenase, a cytoplasmic enzyme formed when Arthrobacter Cr-7 is grown with pyridoxine as a sole source of carbon and nitrogen, was purified about 190-fold to homogeneity from fully induced cells. The enzyme catalyzes Reaction a, (Formula: see text) the essential ring-opening step in the degradation of pyridoxine, and provides a second example of an FAD-dependent oxygenase that adds both two hydrogen and two oxygen atoms to its substrate. 5-Pyridoxic-acid oxygenase has an isoelectric point of 4.6, functions optimally between pH 7 and 8, appears to contain a single subunit of Mr = 51,000 and one FAD (but no iron) per subunit, and is readily resolved by precipitation with ammonium sulfate at pH 3.0. FMN and riboflavin do not replace FAD as coenzyme, but their presence enhances a normally minor side reaction (Reaction b) NAD(P)H + H+ + O2----NAD(P)+ + H2O2 (b) catalyzed by the holoenzyme. Reaction b also is enhanced when the poorly utilized analogues, 3-hydroxy-2-methylpyridine-5-carboxylic acid or NADH, replace 5-pyridoxic acid or NADPH, respectively, as substrates in Reaction a. Each of the enzymes required in two different pathways for degradation of pyridoxine to anabolic intermediates has now been studied. A comparison of these two pathways and their enzymes is provided.
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PMID:Enzymes of vitamin B6 degradation. Purification and properties of 5-pyridoxic-acid oxygenase from Arthrobacter sp. 377 66

Four forms of cellobiose quinone dehydrogenase have been purified from the white-rot fungus Sporotrichum pulverulentum. The Mr of the enzyme has been estimated by sedimentation equilibrium to be 57,800 and by SDS/polyacrylamide-gel to be 60,000. These enzymes are clearly monomers. Cellobiose quinone dehydrogenases contain FAD and variable amounts of a green chromophore which we suggest is 6-hydroxy-FAD. The superoxide anion and H2O2 are the products of its reaction with oxygen. All of the isoenzymes from any one preparation display similar kinetic parameters. However, these vary between preparations. The only apparent difference between the four separable isoenzymes is their neutral-sugar content.
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PMID:Resolution, purification and some properties of the multiple forms of cellobiose quinone dehydrogenase from the white-rot fungus Sporotrichum pulverulentum. 379 72

The FAD binding site of rabbit liver glutathione reductase has been explored by reconstitution of the apoprotein with several FAD analogs modified in the isoalloxazine ring. The apoglutathione reductase binds the p-quinoid form of 8-mercapto-FAD, suggesting that the protein stabilizes a negative charge in the -N1-C2 = O position of the pyrimidine subnucleus. The main absorption peak in the visible spectrum of the 8-mercapto-FAD-enzyme is at 585 nm; treatment of the reconstituted protein with reducing agents of disulfide groups induces a reversible hypochromic shift of 20 nm of the peak. Thus, in 8-mercapto-FAD-glutathione reductase, the oxidation-reduction state of the active center disulfide can be monitored. The chemical reactivity toward methylmethanethiosulfonate and iodoacetamide of the 8-mercapto-FAD-enzyme shows that the flavin position 8 is freely accessible to solvent. However, position 2 is buried within the protein molecule as judged from the lack of reactivity of the 2-thio-FAD-enzyme with methylmethanethiosulfonate. Hydrogen peroxide reacts slowly with both 2-thio-FAD-enzyme and native glutathione reductase, yielding inactive enzyme with a modified spectrum; the prosthetic group is still protein bound. Differences in the active site of the rabbit liver enzyme compared to the human erythrocyte glutathione reductase are evidenced by use of FAD analogs: the peaks of reconstituted liver enzymes are shifted about 10 nm toward longer wavelengths.
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PMID:Properties of rabbit liver glutathione reductase reconstituted with FAD analogs. 394 91


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