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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A membrane-bound NADH dehydrogenase, solubilized and partially purified from a marine bacterium Photobacterium phosphoreum, contains FAD as the prosthetic group, and is specific for NADH. Ferricyanide, various other redox dyes and cytochrome c can act as electron acceptors. The enzymatic activity when assayed with electron acceptors other than cytochrome c, is activated by monovalent cations (Na+ and K+) and deactivated by high concentrations of monovalent anions (SCN-, NO3-, and Cl-) but not by phosphate ions. The enzymatic reaction follows a ping-pong mechanism and kinetic analysis of the enzyme showed that the activation by monovalent cations is due to increase of affinity of the enzyme for substrates; Vm was not affected. The increase of affinity was 62- and 46-fold for NADH and 57- and 31-fold for 2,6-dichlorophenol indophenol in the presence of Na+ and K+, respectively. On the other hand, NADH-cytochrome c reductase activity of the enzyme was strongly inhibited by these cations.
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PMID:Properties and kinetics of salt activation of a membrane-bound NADH dehydrogenase from a marine bacterium Photobacterium phosphoreum. 72 93

8-Thiocyanatoflavins at the riboflavin, FMN, and FAD level were prepared via the diazonium salt of the corresponding 8-aminoflavin and some of the physical and chemical properties studied. 8-Thiocyanatoriboflavin has a UV-visible spectrum similar to that of the native flavin with absorbance maxima at 446 nm (epsilon = 14,900 M-1 cm-1) and 360 nm. Reaction with thiols such as dithiothreitol and mercaptoethanol gives rise to an 8-mercapto- and an 8-SR-flavin, whereas reaction with sulfide yields only the 8-mercaptoflavin. The 8-SCN-flavin binds to riboflavin-binding protein as the riboflavin derivative, to apoflavodoxin, apo-Old Yellow Enzyme, and apo-lactate oxidase as the FMN derivative, and to apo-D-amino acid oxidase, apo-p-hydroxybenzoate hydroxylase, apo-glucose oxidase, apo-anthranilate hydroxylase, and apo-general acyl-CoA dehydrogenase as the FAD derivative. In two cases, namely, with anthranilate hydroxylase and D-amino acid oxidase, the 8-SCN-FAD was spontaneously and completely converted to the 8-mercapto-FAD derivative, suggesting the presence of a nucleophile (most likely the thiol of a cysteine residue) in the vicinity of the 8-position. It was also found that flavodoxin stabilizes the neutral radical and Old Yellow Enzyme the anionic radical of 8-SCN-FMN. Further studies with Old Yellow Enzyme, established that fully (two electron) reduced 8-SCN-FMN undergoes photoelimination of cyanide.
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PMID:8-thiocyanatoflavins as active-site probes for flavoproteins. 167 Sep 91

Recently, the synthesis and properties of several 6-substituted flavins as active site probes for flavoproteins have been reported (Ghisla, S., Massey, V., and Yagi, K. (1986) Biochemistry 25, 3282-3289). Here, we report results of experiments in which 6-thiocyanato-FAD and 6-mercapto-FAD have been substituted for the native flavin of phenol hydroxylase. The 6-SCN-FAD enzyme was converted spontaneously to the 6-mercaptoflavin form probably due to dissociation of flavin, followed by attack of external protein thiols. The pK alpha values of uncomplexed and phenol-bound 6-mercapto-FAD enzyme were determined. Both the spontaneously formed 6-mercapto-FAD enzyme and the enzyme reconstituted with preformed 6-mercapto-FAD were treated with a variety of thiol-specific reagents, and reaction rates were followed by spectroscopic means. Comparison with the corresponding rates found with free flavin suggested a high degree of accessibility to the flavin 6-position. Accessibility was somewhat decreased in the presence of phenol. Upon treatment with low concentrations of methyl methanethiosulfonate or N-ethylmaleimide (NEM), extremely rapid spectral changes were apparent. The former reaction, however, was reversed spontaneously within 2 h. Reaction with NEM was biphasic, with spectral changes consistent with the mechanism previously proposed (Steenkamp, D. J., McIntire, W., and Kenney, W. C. (1978) J. Biol. Chem. 253, 2818-2824), followed by a small absorbance decrease due to protein conformational changes. The NEM reaction is unusual, being easily reversed by addition of excess dithiothreitol.
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PMID:6-Mercapto-FAD and 6-thiocyanato-FAD as active site probes of phenol hydroxylase. 202 45

FAD-modified human glutathione reductases were reconstituted from apoenzyme using the FAD analogues 6-SH-FAD, 6-SCN-FAD, 6-OH-FAD, 6-NH2-FAD and 8-OH-FAD. The catalytic activities of the modified enzymes were substantially lower than for the native enzyme. All five species could be crystallized, but only those containing 6-SH-FAD, 6-OH-FAD and 6-NH2-FAD yielded crystals that could be analyzed. X-ray analyses and structural refinements were performed at 0.27 nm and 0.30 nm resolution resulting in R factors around 13.5%. The crystal structures showed the additional non-hydrogen atoms and small conformational changes of the polypeptide that were obviously induced by the substituents of the FAD analogues. The observed changes together with spectroscopic and activity data permit some conclusions about the chemical nature of the substituents.
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PMID:Structural, spectroscopic and catalytic activity studies on glutathione reductase reconstituted with FAD analogues. 206 68

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

The conformational dynamics of NADH oxidase from Thermus thermophilus was modulated by the Hofmeister series of anions (H2PO4-, SO42-, CH3COO-, Cl-, Br-, I-, ClO4-, SCN-) in the concentration range 0-3 M. Both chaotropic and kosmotropic anions, at high concentration, inhibit the enzyme by different mechanisms. Chaotropic anions increase the apparent Michaelis constant and decrease the activation barrier of the reaction. Kosmotropic anions have the opposite effect. Anions from the middle of the Hofmeister series do not significantly affect the enzyme activity even at high concentration. We detected no significant changes in ellipticity of the aromatic region in the presence of the anions studied. There is a decreased Stern-Volmer quenching constant for FAD fluorescence quenching in the presence of kosmotropic anions and an increased quenching constant in the presence of chaotropic anions. All of this indicates that active site flexibility is important in the function of the enzyme. The data demonstrate that both the high rigidity of the active site in the presence of kosmotropic anions, and its high flexibility in the presence of chaotropic anions have a decelerating effect on enzyme activity. The Hofmeister series of anions proved to be suitable agents for altering enzyme activity through changes in flexibility of the polypeptide chain, with potential importance in modulating extremozyme activity at room temperature.
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PMID:Modulation of activity of NADH oxidase from Thermus thermophilus through change in flexibility in the enzyme active site induced by Hofmeister series anions. 1468 18