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)

Methylenetetrahydrofolate reductase from human cadaver liver was purified to homogeneity. The purified enzyme had a molecular mass of 150 kDa. On SDS-polyacrylamide gel electrophoresis it was dissociated into a single fragment with a molecular mass of 39 kDa. In contrast, fresh lymphocyte enzyme extract showed a major band with a molecular mass of 75 kDa and a minor band of 39 kDa. Fresh liver enzyme was inhibited by S-adenosylmethionine while the purified enzyme from human cadaver liver was not inhibited. These observations suggest that human methylenetetrahydrofolate reductase is composed of two identical subunits of 75 kDa each but is cleaved into a major single band due to autolysis in cadaver liver. The purified cadaver enzyme was a FAD-specific protein. The pH optimum was 6.6 for methylenetetrahydrofolate-NADPH oxidoreductase, 6.5 for methyltetrahydrofolate-menadione oxidoreductase, and 7.2 for NADP-menadione oxidoreductase. The Km values of human liver methylenetetrahydrofolate reductase were 17 microns for NADPH and 38 microns for methyltetrahydrofolate in the reduction of menadione, and 12 microns for NADPH in the reduction of methylenetetrahydrofolate.
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PMID:Purification and characterization of methylenetetrahydrofolate reductase from human cadaver liver. 238 27

Steady-state and laser flash photolysis techniques have been used to examine the photoreduction of yeast glutathione reductase by the one-electron reduction products of 5-deazariboflavin and the viologen analogue 1,1'-propylene-2,2'-bipyridyl. Steady-state photoreduction of the enzyme with the viologen generates the two-electron-reduced form, whereas photoreduction with deazaflavin generates the anion semiquinone. Flash photolysis indicates that the product of viologen radical reduction is also a semiquinone, suggesting that this species is rapidly further reduced by viologen in the steady-state experiment to form the EH2 enzyme. This reduction is apparently inhibited when deazaflavin is the photoreductant, perhaps due to complexation of the anion semiquinone with deazaflavin. Steady-state experiments demonstrate that complexation of the anion semiquinone with NADP+ also inhibits further reduction. Both one-electron reduction reactions of oxidized glutathione reductase proceed at close to diffusion-controlled rates (second-order rate constants = 10(8)-10(9) M-1 s-1), despite the relatively buried nature of the FAD cofactor. Addition of NADP+ and oxidized glutathione produced no effects on the kinetics of the initial entry of the electron into the enzyme. No kinetic evidence of intramolecular electron transfer involving the FAD and the protein disulfide was obtained during or subsequent to the initial one-electron reduction process. Thus, if this reaction occurs in the semiquinone, it must be quite rapid (k greater than 8000 s-1).
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PMID:Steady-state and laser flash induced photoreduction of yeast glutathione reductase by 5-deazariboflavin and by a viologen analogue: stabilization of flavin adenine dinucleotide semiquinone species by complexation. 238 72

The electrostatically stabilized complex between Anabaena variabilis ferredoxin--NADP+ reductase and Azotobacter vinelandii flavodoxin has been covalently cross-linked by treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex exhibits a molecular mass and FMN/FAD content consistent with that expected for a 1:1 stoichiometry of the two flavoproteins. Immunochemical cross-reactivity is exhibited by the covalent complex with rabbit antisera prepared separately against each protein. The complex retains NADPH-ferricyanide diaphorase activity although the Km for ferricyanide is increased twofold and the turnover number is decreased by a factor of two when compared to native reductase. NADPH-cytochrome-c reductase activity of the complex is observed at a level that is quite similar to that determined at saturating concentrations of flavodoxin, while it is only 1-2% of that exhibited by the reductase in the presence of ferredoxin. No stimulation of cytochrome-c reductase activity is observed on adding ferredoxin to the cross-linked complex. Stopped-flow data show that covalent cross-linking of the flavodoxin to the reductase reduces the rate of electron transfer from its semiquinone form to cytochrome c by a factor of 60. Anaerobic titrations of the reduced complex with NADP+ show the semiquinone/quinol couple of the flavodoxin is increased 100 mV relative to the free form and the quinone/quinol couple of complexed ferredoxin-NADP+ reductase is increased by only 25 mV, relative to the free protein. Addition of NADPH to the cross-linked complex reduces the FAD of the reductase as well as the FMN moiety of flavodoxin to a mixture of semiquinone and quinol forms.
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PMID:Preparation and properties of a cross-linked complex between ferredoxin--NADP+ reductase and flavodoxin. 250 11

During the past 15 years, the development of strategies to apply the catalytic potential of redox coenzyme-requiring enzymes has been a subject of intensive study; the main purpose of which has been to cut the cost of coenzyme to an economically acceptable level. One approach has been the utilization of isolated coenzyme-dependent enzyme systems with simultaneous enzymatic coenzyme regeneration (recycling). This has been used in conjugation with ultrafiltration reactor technology (enzyme membrane reactor), with coenzyme concentration being kept at a catalytic level. The concept implies confinement (immobilization) and practically 100% retention of both enzymes and coenzymes being dissolved in homogeneous solution within the reactor space that is closed off by an ultrafiltration membrane through which low-molecular-weight reactants (substrates and products) can freely pass. Since the problem of retaining nearly 100% native coenzymes of relatively low molecular weight by ultrafiltration membranes has not been satisfactorily solved, active macromolecular coenzyme derivatives are required. In this review, the syntheses, properties and merits of water-soluble macromolecular derivatives of NAD(H), NADP(H) and FAD are considered with respect to their biotechnological application.
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PMID:Synthesis and application of water-soluble macromolecular derivatives of the redox coenzymes NAD(H), NADP(H) and FAD. 251 Apr 75

Mercuric ion reductase (the merA gene product) is a unique member of the class of FAD and redox-active disulfide-containing oxidoreductases by virtue of its ability to reduce Hg(II) to Hg(0) as the last step in bacterial detoxification of mercurials. In addition to the active site redox-active disulfide, formed between Cys135 and Cys140 in Tn501 MerA, the protein products of the three merA gene sequences published to date have two additional conserved pairs of cysteines, one near the N-terminus (Cys10Cys13 in Tn501 MerA) and another near the C-terminus (Cys558Cys559 in Tn501 MerA). Neither of these pairs is found in other members of this enzyme family. To assess the possible roles of these peripheral cysteines in the Hg(II) detoxification pathway, we have constructed and characterized one single mutant, Cys10Ala13, and two double mutants, Ala10Ala13 and Ala558Ala559. The N-terminal mutants are fully functional in vivo as determined by HgCl2 resistance studies, showing the N-terminal cysteine pair to be dispensable. In contrast, the Ala558Ala559 mutant is defective for HgCl2 resistance in vivo and Hg(SR)2 reduction in vitro, thereby implicating Cys558 and/or Cys559 in Hg(II) reduction by the wild-type enzyme. Other activities, such as NADPH/thio-NADP+ transhydrogenation, NADPH oxidation, and DTNB reduction, are unimpaired in this mutant.
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PMID:Mutagenesis of the N- and C-terminal cysteine pairs of Tn501 mercuric ion reductase: consequences for bacterial detoxification of mercurials. 254 Aug 17

In dialyzed bovine brain cytosol, the enzymatic formation of nitrogen oxides was directly determined. The basal formation of nitrite and nitrate was concentration-dependently enhanced by L-arginine (EC50 about 3.10(-5) M). Both the basal and L-arginine induced formations were inhibited by NG-monomethyl-L-arginine (EC50 about 2.10(-4) M). In the presence of L-arginine, a concomitant formation of citrulline was detected. L-Arginine methyl ester also served as a substrate, but neither D-arginine, D-arginine methyl ester nor N alpha-benzoyl-L-arginine ethyl ester did so. The formation of nitrite and nitrate was time-dependent, increased linearly with the protein concentration of the cytosol and was not observed when the cytosolic proteins were heat-denaturated. Exogenous NADPH (or NADP+) concentration-dependently enhanced the formation of nitrite and nitrate, whereas NADH, NAD+, FAD, Ca2+, Mg2+ and calmodulin were ineffective. These results indicate that bovine brain contains a cytosolic enzyme which uses NADPH or NADP+ as cofactors to form nitrogen oxides from both an endogenous non-dialyzable substrate and from L-arginine.
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PMID:Enzymatic formation of nitrogen oxides from L-arginine in bovine brain cytosol. 259 Feb 27

Yeast glutathione reductase (E.C. 1.6.4.2) catalyzes the oxidation of NADPH by p-quinones and ferricyanide with a maximal turnover number (TNmax) of 4-5 s-1.NADP+ stimulates the reaction and the TNmax/Km value of acceptors is reached at NADP+/NADPH greater than or equal to 100. TNmax is increased up to 30-33 s-1. The stimulatory effect of NADP+ may be associated with its complexation with the NADPH-binding site in the reduced enzyme (Kd = 40-60 microM). It is suggested that NADP+ shifts the electron density towards FAD in the two-electron-reduced enzyme and, evidently, changes its one-electron-reduction potentials, while quinones oxidize an equilibrium form of glutathione reductase containing reduced FAD. In the absence of NADP+ the reduction of quinones by glutathione reductase proceeds mainly in a two-electron manner. At NADP+/NADPH = 100 a one-electron reduction makes up 44% of the total process. At pH 6.0-7.0 the reduced forms of naphthoquinones undergo cyclic redox conversions. A hyperbolic dependence exists of the log TN/Km of quinones on their one-electron-reduction potentials.
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PMID:One- and two-electron reduction of quinones by glutathione reductase. 264 41

Glutathione reductase from Escherichia coli is inactivated when incubated with either NADPH or NADH. The process is inversely dependent on the enzyme concentration. Inactivation is rapid and monophasic with 1 microM NADPH and 1 nM enzyme FAD giving a t1/2 of 1 min. Complex formation between NADPH and the two-electron reduced enzyme (EH2) at higher levels of NADPH protects against rapid inactivation. NADP+, produced in a side reaction with oxygen, also protects by forming a complex with EH2. These complexes make analysis of the concentration dependence of the inactivation process difficult. Inactivation with NADH, where complexes do not interfere, is slower but can be analyzed more readily. With 152 microM NADH and 5.4 nM enzyme FAD, the time required for 50% inactivation is 17 min. The process is markedly biphasic, reaching the final inactivation level after 5-7 h. Analysis of the relationship between the final level of inactivation with NADH and the enzyme concentration indicates that inactivation is due to dissociation of the normally dimeric enzyme. Thus, the position of the dimer-monomer equilibrium between an active dimeric two-electron reduced species and an inactive monomeric two-electron reduced form determines the enzyme activity. An apparent equilibrium constant (Kd) for dissociation of dimer obtained from the anaerobic concentration dependent inactivation curves is 220 nM. Enzyme inactivated with NADH can be reactivated with glutathione, and the reactivation kinetics are second order, monomer-monomer over 75% of the reaction with an average apparent association rate constant (ka) of 13.1 (+/- 5.5) X 10(6) M-1 min-1.
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PMID:Inactivation-reactivation of two-electron reduced Escherichia coli glutathione reductase involving a dimer-monomer equilibrium. 266 73

Glutathione reductase from S. cerevisiae (EC 1.6.4.2) catalyzes the NADPH oxidation by glutathione in accordance with a "ping-pong" scheme. The catalytic constant kcat) is 240 s-1 (pH 7.0, 25 degrees C); kcat for the diaphorase reaction is 4-5 s-1. The enzyme activity does not change markedly at pH 5.5-8.0. At pH less than or equal to 7.0, NADP+ acts as a competitive inhibitor towards NADPH and as a noncompetitive inhibitor towards glutathione. NADP+ increases the diaphorase activity of the enzyme. The maximal activity is observed, when the NADP+/NADPH ratio exceeds 100. At pH 8.0, NADP+ acts as a mixed type inhibitor during the reduction of glutathione. High concentrations of NADP+ also inhibit the diaphorase activity due to the reoxidation of the reduced enzyme by NADP+ at pH 8.0. The redox potential of glutathione reductase calculated from the inhibition data is--306 mV (pH 8.0). Glutathione reductase reduces quinoidal compounds in an one-electron way. The hyperbolic dependence of the logarithm of the oxidation constant on the one electron reduction potential of quinone is observed. It is assumed that quinones oxidize the equilibtium fraction of the two-electron reduced enzyme containing reduced FAD.
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PMID:[The relation of glutathione reductase and diaphorase activity of glutathione reductase from Saccharomyces cerevisiae]. 267 96

Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases [acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester hydrolyzing), EC 2.3.1.85] and yeast fatty acid synthase [fatty-acyl-CoA synthase; acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing), EC 2.3.1.86] were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chicken and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The NADPH-binding dinucleotide folds of the beta-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the NADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution. A higher mutation rate in the joining regions than in the active site regions of the enzymes without loss of function might be expected.
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PMID:Homology analysis of the protein sequences of fatty acid synthases from chicken liver, rat mammary gland, and yeast. 268 49


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