Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 effect of nicotinamide and flavin coenzymes on the 5-lipoxygenase activity has been determined in cell-free extracts from rat polymorphonuclear leukocytes. 5-lipoxygenase was assayed in the presence of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which caused a 3 to 4-fold stimulation in the maximal conversion of radiolabeled arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE) and 5,12-dihydroxyeicosatetraenoic acid (5,12-di-HETE). Addition of FMN or FAD to the assay mixture had little effect on the 5-lipoxygenase activity and caused inhibition only at high concentrations (IC50 greater than 100 microM). NADH markedly potentiated the inhibition of lipoxygenase by flavins with a 100-fold decrease in the FMN concentration required to inhibit the enzyme (IC50 approximately equal to 2 microM). Similar effects were observed for FAD although this flavin derivative was slightly less potent than FMN (IC50 congruent to 10 microM). NADH could be substituted by NADPH but not by NAD or NADP, indicating that the inhibition was not due to the production of the oxidized forms of these co-factors. These results show that the 5-lipoxygenase activity is stimulated by 5-HPETE and inhibited by flavin-dependent redox transformations.
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PMID:Modulation of rat polymorphonuclear leukocyte 5-lipoxygenase activity by 5-HPETE and NADH-dependent flavin inhibition. 309 90

Beef liver and human erythrocyte catalases (EC 1.11.1.6) bind NADP tenaciously [Kirkman, H. N. & Gaetani, G. F. (1984) Proc. Natl. Acad. Sci. USA 81, 4343-4348]. The position of NADP on beef liver catalase corresponds to the carboxyl-terminal polypeptide hinge in Penicillium vitale fungal catalase, which connects the common catalase structure to the additional flavodoxin-like domain. In contrast to nearly all other known structures of protein-bound NADP, NAD, and FAD, the NADP molecule of beef liver catalase is folded into a right-handed helix and bound, in part, in the vicinity of the carboxyl end of two alpha-helices. A water molecule (W7) occupies a pseudosubstrate site close to the C4 position of the nicotinamide and is hydrogen bonded to His-304. Although the NADP and heme groups approach each other to within 13.7 A, there is no direct interaction. The function of the NADP remains a mystery.
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PMID:The NADPH binding site on beef liver catalase. 385 39

The reduction of yeast glutathione reductase by reduced nicotinamide hypoxanthine dinucleotide phosphate (NHxDPH) has been examined by stopped-flow kinetic methods. Like reduced glutathione or NADPH, this pyridine nucleotide generates the catalytically active two-electron reduced form of the enzyme. This reductive half-reaction with NHxDPH has only one detectable kinetic step which shows saturation kinetics (Kd = 76 microM), and has a limiting rate constant of 56 s-1. Comparison of stopped-flow and steady-state turnover data indicates that the reductive half-reaction is rate-limiting in the overall catalytic reaction. No evidence was found for a preequilibrium charge-transfer complex between NHxDPH and the active site FAD, like that seen when NADPH is the electron donor.
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PMID:Kinetic studies of the reduction of yeast glutathione reductase by reduced nicotinamide hypoxanthine dinucleotide phosphate. 388 56

1. The l-malate dehydrogenase of Pseudomonas ovalis Chester, which is independent of nicotinamide nucleotides and which is structurally and functionally bound to the cell-wall membrane, has been prepared in a soluble form and partially purified. 2. The purified dehydrogenase exhibits a triple cofactor requirement for FAD, quinone and phospholipid, and in the presence of these cofactors can utilize 2,6-dichlorophenol-indophenol as hydrogen acceptor. 3. The formation of reduced forms of FAD was not detected, but in the presence of both FAD and phospholipid the enzyme catalysed the reduction of quinone by l-malate at rates equivalent to those obtained with 2,6-dichlorophenol-indophenol as terminal acceptor. The l-malate dehydrogenase of Ps. ovalis Chester is therefore an l-malate-quinone oxidoreductase. 4. The quinone and the phospholipids present in the fragments of the cell-wall membrane from which the soluble dehydrogenase was prepared have been extracted and purified. The quinone was identified as coenzyme Q(9). At least eight phospholipids were detected, and the major component is an unsaturated phosphatidylethanolamine. 5. The nature of the phospholipid required to activate the enzyme depends on the nature of the quinone used in the assay system. When 2-methyl-1,4-naphthaquinone is used, a wide variety of phospholipids, including all those isolated from the organism, will activate the enzyme, but when coenzyme Q(9) is used the phospholipid specificity of the enzyme is much more restricted, and the most effective activator is the unsaturated phosphatidylethanolamine isolated from the organism. 6. Evidence is presented to support the view that the restricted phospholipid specificity exhibited by the enzyme in the presence of coenzyme Q(9), as opposed to the broad specificity exhibited when 2-methyl-1,4-naphthaquinone is used, is due to the fact that coenzyme Q(9) has a large substituent on position 3.
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PMID:Cofactor requirements of the L-malate dehydrogenase of Pseudomonas ovalis Chester. 596 84

NAD (P) H-dependent reduction of nicotinamide N-oxide was investigated with rabbit liver preparations. Microsomes, microsomal NADPH-cytochrome c reductase or cytosolic aldehyde oxidase alone exhibited no nicotinamide N-oxide reductase activity in the presence of NADPH or NADH. However, when the microsomal preparations were combined with the cytosolic enzyme, a significant N-oxide reductase activity was observed in the presence of the reduced pyridine nucleotide. The activity was enhanced by FAD or methyl viologen. Cytosol alone supplemented with NADPH or NADH exhibited only a slight, but when combined with microsomes, a significant N-oxide reductase activity. Based on these facts, we propose a new electron transfer system consisting of NADPH-cytochrome c reductase and aldehyde oxidase, which exhibits nicotinamide N-oxide reductase activity in the presence of the reduced pyridine nucleotide.
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PMID:NAD (P) H-dependent reduction of nicotinamide N-oxide by an unique enzyme system consisting of liver microsomal NADPH-cytochrome C reductase and cytosolic aldehyde oxidase. 624 Feb 69

The mode of binding of NADPH and oxidized glutathione to the flavoenzyme glutathione reductase has been determined by x-ray crystallography. Furthermore, two intermediates of the reaction have been produced in the crystal and have been structurally elucidated. All these analyses were done at 0.3 nm resolution. The results allow the stereochemical description of the mechanism of the enzyme. The dinucleotide NADPH is bound in an extended conformation with the nicotinamide ring stacking onto the re-face of the flavin part of FAD, and adenine located at the protein surface. The binding of NADPH results in the 2-electron reduced form of the enzyme, EH2. This form has also been analyzed without any ligand bound. In EH2 the redoxactive disulfide bridge of the protein, which lies at the si-face of the flavin ring, is opened and the sulfur of Cys-58 moves by about 0.1 nm into a position where it can attack one of the sulfurs of the substrate oxidized glutathione. This interchange leads to a mixed glutathione-protein disulfide, which can be stabilized in crystals and has been analyzed. By selectively reacting Cys-58 with iodoacetamide the crystalline enzyme can be blocked in its EH2 state. The imidazole of His-467' is near to all sulfurs taking part in the disulfide bridge exchange and is therefore certainly crucial for catalysis. The crystallographic results establish that electrons flow from NADPH to the substrate GSSG via flavin and the redoxactive protein disulfide bridge. This is consistent with the scheme that has been postulated from biochemical, spectroscopic, and model studies.
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PMID:The catalytic mechanism of glutathione reductase as derived from x-ray diffraction analyses of reaction intermediates. 682 32

Trypanothione reductase is a member of the structurally and functionally well-characterized family of flavoprotein reductases, which catalyze the reduced pyridine nucleotide dependent reduction of their disulfide, peroxide, or metal ion substrates. Trypanothione reductase is found in a wide variety of Trypanosoma species, where the enzyme serves physiologically to protect the organism from oxidative stress and assists in maintaining low intracellular levels of hydrogen peroxide. The redox potential of the flavin and the hydride ion transfer reaction of the pro-S hydrogen of NADPH to N5 of FAD have been proposed to be influenced by the presence of a conserved Lys-Glu (K60-E201) ion pair at the bottom of the nicotinamide binding pocket. We have evaluated this hypothesis by making modest substitutions for both the Lys and Glu residues using site-directed mutagenesis. Replacement of the K60 residue with an arginine led to a poorly expressed, and completely inactive, enzyme. Replacement of the Glu201 residue with either a glutamine (E201Q) or an aspartate (E201D) residue led to expressed enzymes which could be readily purified in > 20 mg amounts using protocols developed for the WT enzyme, and which had significant residual trypanothione-reducing activity. These enzymes have now been characterized to determine their redox potentials, catalytic activities, and nucleotide specificities. Relative to the WT enzyme, both E201D and E201Q exhibit ca. 5% of WT trypanothione-reducing activity using NADPH as reductant, but significantly enhanced quinone reductase activity. The oxidase activity of both mutants is enhanced by over 50-fold compared to that of the WT. The redox potential of the WT enzyme has been determined to be -273 mV, while both the E201D and E201Q exhibit more positive redox potentials (-259 and -251 mV, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catalytic and potentiometric characterization of E201D and E201Q mutants of Trypanosoma congolense trypanothione reductase. 754 22

The contribution made by tyrosine 308 to the stability of pea ferredoxin-NADP+ reductase was investigated using site-directed mutagenesis. The phenol side chain of the invariant carboxyl terminal tyrosine is stacked coplanar to the isoalloxazine moiety of the FAD cofactor. Fluorescence measurements indicate that this interaction plays a significant role in FAD fluorescent quenching by the reductase apoprotein. Replacement of the tyrosine by tryptophan or phenylalanine caused only a minor increase in the quantum yields of bound FAD, whereas nonaromatic substitutions to serine and glycine resulted in a large fluorescent rise. Results from NADP+ titration experiments support a recent hypothesis [Karplus et al. (1991) Science 251, 60-66], suggesting that the phenol ring of Tyr 308 may fill the nicotinamide binding pocket in the absence of the nucleotide. The stability of the site-directed mutants, judged by thermal- and urea-induced denaturation studies, was lowered with respect to the wild-type enzyme. FNR variants harboring nonaromatic substitutions displayed more extensive destabilization. The decrease in thermodynamic stability correlated with the impairment of catalytic activities [Orellano et al. (1993) J. Biol. Chem 268, 19267-19273]. The results indicate that the presence of the electron-rich aromatic side chain adjacent to the isoalloxazine ring is essential for maximum stabilization of the FNR holoenzyme, resulting in a flavin conformation which optimizes electron flow between the prosthetic group and its redox partners.
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PMID:Contribution of the FAD binding site residue tyrosine 308 to the stability of pea ferredoxin-NADP+ oxidoreductase. 754 39

The flavoprotein thioredoxin reductase catalyzes the reduction of the small redox protein thioredoxin by NADPH. Thioredoxin reductase contains a redox active disulfide and is a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes that includes lipoamide dehydrogenase, glutathione reductase, trypanothione reductase, mercuric reductase, and NADH peroxidase. The structure of thioredoxin reductase has recently been determined from X-ray crystallographic data. In this paper, we attempt to correlate the structure with a considerable body of mechanistic data and to arrive at a mechanism consistent with both. The path of reducing equivalents in catalysis by glutathione reductase and lipoamide dehydrogenase is clear. To envisage the path of reducing equivalents in catalysis by thioredoxin reductase, a conformational change is required in which the NADPH domain rotates relative to the FAD domain. The rotation moves the nascent dithiol from its observed position adjacent to the re surface of the flavin ring system toward the protein surface for dithiol-disulfide interchange with the protein substrate thioredoxin and moves the nicotinamide ring of NADPH adjacent to the flavin ring for efficient hydride transfer. Reverse rotation allows reduction of the redox active disulfide by the reduced flavin. This requires that the enzyme pass through a ternary complex; the kinetic evidence for such a complex is discussed.
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PMID:Mechanism and structure of thioredoxin reductase from Escherichia coli. 755 16

The crystal structure of ferredoxin-NADP+ reductase (FNR) suggests that Ser96 is directly involved in hydride transfer between the isoalloxazine moiety of FAD and the nicotinamide ring of NADP(H). To probe its role, Ser96 has been mutated to valine (S96V) and glycine (S96G). These mutations primarily affected the interaction of the nicotinamide ring with the flavin. Absorbance, fluorescence, and circular dichroism spectra and the crystal structure of FNR-S96V indicate that this mutant folds properly. FNR-S96V shows only 0.05% of wild-type activity, while the affinities for both ferredoxin and NADP+ are virtually unchanged. However, spectral perturbations induced by NADP+ binding to FNR-S96V strongly resemble those elicited by the binding of 2'-monophosphoadenosine-5'-diphosphoribose, a substrate analog lacking the nicotinamide ring, both to the mutant and wild-type enzymes. Rapid reaction studies on the valine mutant failed to detect charge-transfer intermediates during flavin reduction by NADPH. In addition, no semiquinone formation was seen during photoreduction of FNR-S96V. The three-dimensional structure of the valine mutant shows small, albeit definite, changes only in the isoalloxazine microenvironment. The glycine mutant of FNR displays behavior intermediate between that of wild-type enzyme and that of the valine mutant. It maintains ca. 2% of the wild-type activity as well as the ability to form the charge-transfer species between reduced FNR and NADP+. In photoreduction experiments, the same degree of flavin semiquinone stabilization was observed with FNR-S96G and with the wild-type enzyme. NADP+ binding to the glycine mutant was very similar to that observed in the case of the valine mutant.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Involvement of serine 96 in the catalytic mechanism of ferredoxin-NADP+ reductase: structure--function relationship as studied by site-directed mutagenesis and X-ray crystallography. 767 50


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