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: EC:1.6.5.2 (NQO1)
6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Diethyl pyrocarbonate inhibited diaphorase activity of ferredoxin-NADP+ oxidoreductase with a second-order rate constant of 2 mM-1 X min-1 at pH 7.0 and 20 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity could be restored by hydroxylamine, and the pH curve of inactivation indicated the involvement of a residue having a pKa of 6.8. Derivatization of tyrosyl residues was also evident, although with no effect on the diaphorase activity. Both NADP+ and NADPH protected the enzyme against inactivation, suggesting that the modification occurred at or near the nucleotide binding domain. The reductase lost all of its diaphorase activity after about two histidine residues had been blocked by the reagent. In differential-labeling experiments with NADP+ as protective agent, it was shown that diaphorase inactivation resulted from blocking of only one histidyl residue per mole of enzyme. Modified reductase did not bind pyridine nucleotides. Modification of the flavoprotein in the presence of NADP+, i.e., with full preservation of diaphorase activity, resulted in a significant impairment of cytochrome c reductase activity, with a second-order rate constant for inactivation of about 0.5 mM-1 X min-1. Reversal by hydroxylamine and spectroscopic data indicated that this second residue was also a histidine. Ferredoxin afforded only slight protection against this inhibition. Conversely, carbethoxylation of the enzyme did not affect complex formation with the ferrosulfoprotein. Redox titration of the modified reductase with NADPH and with reduced ferredoxin suggested that the second histidine might be located in the electron pathway between FAD and ferredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Essential histidyl residues of ferredoxin-NADP+ oxidoreductase revealed by diethyl pyrocarbonate inactivation. 668 70

NADPH-dependent O2- -generating activity was extracted and partially purified from guinea pig polymorphonuclear leukocytes. The most active preparation generated 202.8 nmol O2- min/min per mg protein. This activity was 30-fold higher than that of extracts from resting cells, indicating that the activated state of the oxidase was retained after solubilization. The solubilization and purification of the enzyme activity were followed by a parallel solubilization and purification of cytochrome b. Spectroscopic studies showed that solubilized cytochrome b has an Em of -245 mV and binds CO to about 30%. Cytochrome b was reduced by NADPH in anaerobiosis at a low rate and was rapidly reoxidized by air. A correlation was found between the inhibition of O2- formation caused by the SH reagent p-chloromercuribenzoate and the alterations induced by this compound on the Em of cytochrome b. These observations strongly support the participation of cytochrome b in the catalytic activity of the solubilized NADPH oxidase. The enzyme preparations contained FAD, which was found to be associated both with NADPH oxidase and with diaphorase activities. The fraction with the highest O2- forming activity contained FAD and cytochrome b in a ratio of about 0.5:1. The participation of FAD in the electron transport from NADPH to O2 is supported also by the inhibitory effect exerted by quinacrine on O2- formation.
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PMID:The cytochrome b and flavin content and properties of the O2- -forming NADPH oxidase solubilized from activated neutrophils. 687 Dec 31

A human FAD-dependent diaphorase, DIA4, has been studied in 29 independent human-rodent hybrids and in 17 subclones. The results suggest that the locus DIA4 is on chromosome 16.
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PMID:Assignment of a human diaphorase (DIA4) to chromosome 16. 689 12

The purification and properties of metlegoglobin reductase from lupine (Lupinus luteus L.) nodules are described. The purification procedure results in a 1056-fold purification of the enzyme with a total yield of 21%. The enzyme possesses the NADH-diaphorase activity. Metlegoglobin reductase is heterogenous during electrophoresis and isoelectric focusing. Electrophoresis produces two vicinal active bands, while isoelectrofocusing results in four active fractions. The fraction possessing the highest activity has a pI of 4.4. The enzyme is a flavoprotein, in which all flavins are represented by FAD. The molecular weight of the enzyme is 30 000. In some properties metlegoglobin reductase from lupine nodules is similar to methemoglobin reductase from erythrocytes and metmyoglobin reductase from muscles.
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PMID:[Properties of metlegoglobin reductase from lupine nodules]. 689 54

The purification by affinity chromatography up to homogeneity and the properties of NAD-reductase from purple sulfur bacterium Thiocapsa roseopersicina, strain BBS, are described. The molecular weight of NAD-reductase is about 80000; pI is 3.9. The enzyme consists of two subunits. According to the stabilizing effect of FAD at preparative electrophoresis and the inhibitory effect of atebrine NAD-reductase is a flavoprotein. The bulk of the enzyme (about 75%) is localized in the cell periplasmic space. NAD-reductase is less thermostable and has a lower O2 stability as compared to the NADP-reductase from the same organism. The enzyme is specific to NADH ane catalyzes the menadione-reductase reaction, diaphorase reaction of benzyl viologen and methyl viologen reductions. In the presence of NADH NAD-reductase reduces cytochromes c552 and "c3" from T. roseopersicina and forms a complex with spinach ferredoxin.
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PMID:[Purification and properties of NAD-reductase from phototrophic bacterium Thiocapsa roseopersicina]. 723 99

D-Lactate dehydrogenase, the starting enzyme for carbon and energy metabolism in dissimilatory sulfate-reducing bacteria, has been purified 36-fold from the soluble fraction of the sonicate of Desulfovibrio vulgaris, Miyazaki. The enzyme is specific for D-lactate (Km = 0.8 mM) and DL-2-hydroxybutyrate (probably its D-isomer) as the electron donor substrate. It reduces, in the presence of lactate, various artificial electron acceptors such as 1-methoxyphenazinium methyl sulfate, ferricyanide, tetrazolium dyes, methylene blue, and 2,6-dichlorophenol-indophenol. When 2 mol of ferricyanide was reduced, 1 mol of pyruvate was produced during the reaction. Among natural electron carriers, only cytochrome c-553 isolated from the same organism can be reduced by the enzyme. The ferric complex of pyridine-2,6-dicarboxylate can act as an electron acceptor if cytochrome c-553 is present in the reaction system. NAD+, NADP+, FAD, FMN, cytochrome c3, high-molecular-weight cytochrome, eucaryotic cytochromes c (yeast and horse) and O2 could not be reduced. The enzyme does not have any diaphorase activity. The D-lactate dehydrogenase of D. vulgaris must therefore be named D-lactate:ferricytochrome c-553 oxidoreductase [EC subclass 1.1.2]. A similar enzyme exists in the formate dehydrogenase-less mutant of D. vulgaris, Miyazaki, and in D. vulgaris, Hildenborough.
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PMID:D-lactate dehydrogenase of Desulfovibrio vulgaris. 727 46

A newly discovered human diaphorase, designated diaphorase-4, which accounts for a major part of the diaphorase activity of most tissues but does not occur in erythrocytes, is described. In contrast with other human diaphorases, it is dependent on FAD for activity after electrophoresis, inhibited by low concentrations of dicoumarol and shows a marked affinity for Cibacron Blue. The molecular weight was estimated to be 49000 +/- 1800 by gel filtration. Diaphorase-4 appears to show person-to-person quantitative variation, so that about 4% of the population lack appreciable enzyme activity, but it is not yet clear whether this variation is of genetic or non-genetic origin.
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PMID:Human FAD-dependent NAD(P)H diaphorase. 739 57

NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is a FAD-containing reductase that catalyzes a unique 2-electron reduction of quinones. It consists of 2 identical subunits. In this study, it was found that the carboxyl-terminal portion of the 2 subunits can be cleaved by various proteases, whereas the amino-terminal portion cannot. It was also found that proteolytic digestion of the enzyme can be blocked by the prosthetic group FAD, substrates NAD(P)H and menadione, and inhibitors dicoumarol and phenindione. Interestingly, chrysin and Cibacron blue, 2 additional inhibitors, cannot protect the enzyme from proteolytic digestion. The results obtained from this study indicate that the subunit of the quinone reductase has a 2-domain structure, i.e., an amino-terminal compact domain and a carboxyl-terminal flexible domain. A structural model of the quinone reductase is generated based on results obtained from amino-terminal and carboxyl-terminal protein sequence analyses and electrospray mass spectral analyses of hydrolytic products of the enzyme generated by trypsin, chymotrypsin, and Staphylococcus aureus protease. Furthermore, based on the data, it is suggested that the binding of substrates involves an interaction between 2 structural domains.
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PMID:A two-domain structure for the two subunits of NAD(P)H:quinone acceptor oxidoreductase. 751 54

NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is an FAD-containing enzyme that catalyzes the 2-electron reduction of quinones to hydroquinones using either NADH or NADPH as the electron donor. In this study, FAD was removed by dialyzing the holoprotein against 2 M KBr, and synthetic analogs of FAD were substituted in the flavin binding site as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-mercapto-FAD are stabilized on binding to the enzyme. This is consistent with the fact that the native flavoprotein forms the anion flavin radical upon photoreduction and suggests the presence of a positive charge near the N(1)C(2)O position of the isoalloxazine ring. Reactivity studies using 8-chloro- and 8-mercapto-flavins suggest that the 8 position of the FAD is accessible to the solvent. However, the rates of the reactions were dramatically decreased in the presence of the competitive inhibitor, dicumarol. 6-Mercapto-, 6-thiocyanato-, 6-azido-, and 6-amino-flavins were also used as structural probes. The results indicate that the 6 position is accessible to solvent. Dicumarol binding increases the pK alpha of the enzyme-bound 6-mercapto-flavin from below pH 5.0 to higher than pH 9.0. The results suggest that DT-diaphorase shows the same properties as the C-C transhydrogenases, and the binding of dicumarol elicits a conformational change or an adjustment in the polarity of the FAD pocket. The enzyme reconstituted with oxidized 5-deaza-FAD has significant catalytic activity, confirming that DT-diaphorase is an obligatory 2-electron transfer enzyme and plays a role in the detoxification of quinones and quinoid compounds by reducing them to the relatively stable hydroquinones.
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PMID:Active site studies of DT-diaphorase employing artificial flavins. 753 91

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


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