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)

The leukocyte iodonitrotetrazolium violet (INT) reductase activity of disrupted bovine polymorphonuclear neutrophils is closely associated with the activation of the O2(-)-generating NADPH oxidase in a cell-free system. It is dependent upon NADPH, cytosolic factors, and amphiphiles (such as arachidonate), the same factors required for O2- generation. Both O2- generation and INT reductase activity are inhibited by phenylarsine oxide, an inhibitor of the activation of the NADPH oxidase [Li, J., & Guillory, R. J. (1997) J. Biochem. Mol. Biol. Biophys. (in press)]. In this report, the INT diaphorase activity of disrupted bovine polymorphonuclear neutrophils is shown to be resolved by DEAE-Sepharose chromatography into two fractions: an NADPH-cytochrome c reductase-containing fraction and a cytochrome b558-associated fraction. The diaphorase activity in the NADPH-cytochrome c reductase-containing portion is not dependent upon the presence of an amphiphile or phospholipid and is not associated with O2- generation. Upon incorporation into liposomes, the cytochrome b558-containing fraction demonstrates high O2- and INT reductase activities in the presence of cytosolic factors. Both O2- generation and INT reductase activities are SDS and FAD dependent and further stimulated by GTPgammaS. Phenylarsine oxide inhibits both O2- generation and INT reductase activities when added prior to activation by SDS. With the cytochrome b-containing liposomes, the Km values (O2- formation) for NADPH and NADH are 27.2 microM and 810 microM, and for INT reductase the Km values are 27.5 microM and 1017 microM, respectively. Under anaerobic conditions and thus in the absence of O2- formation, the NADPH-dependent INT reductase activity does not change, indicating that the dye reduction is not due to its direct reduction by O2 anion but is an intrinsic property of the superoxide-generating NADPH oxidase. Cytochrome b558 is the essential component of the NADPH oxidase and contains all the redox centers necessary for electron flow between NADPH and oxygen. The correlation of the activation and inhibition patterns for O2- generation and INT reduction by cytochrome b558 incorporated into artificial liposomes strongly indicates that the two activities are associated with the same membrane protein, cytochrome b558.
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PMID:Purified leukocyte cytochrome b558 incorporated into liposomes catalyzes a cytosolic factor dependent diaphorase activity. 915 36

The quaternary behaviour of DT diaphorase in solution has been investigated by hydrodynamics under a range of conditions. At neutral pH DT diaphorase is shown to exist as a tightly-associated homodimer in a dimer-tetramer equilibrium. Concentrations of the chaotropic agent potassium thiocyanate (KSCN) of greater than 200 mM result in irreversible loss of the FAD cofactor and denaturation of the homodimer though this agent appears to be ineffective in disrupting intermolecular association. These data conform to a model in which under extreme dissociation conditions, the folded dimer is in equilibrium with the unfolded monomer and are consistent with evidence from the X-ray structure and proposed catalytic mechanism where both monomers are catalytically interdependent.
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PMID:DT diaphorase exists as a dimer-tetramer equilibrium in solution. 918 64

The assimilatory nitrate reductase from the phototrophic bacterium Rhodobacter capsulatus has been purified to electrophoretic homogeneity and its molecular and kinetic parameters determined. The native nitrate reductase is a dimer of 144 kDa composed of two subunits of 46 and 95 kDa. The purified enzyme catalyzes the electron transfer from NADH, reduced bromophenol blue or reduced viologens to nitrate. The nitrate reductase contains 1 mol FAD per mole of enzyme and also reduces cytochrome c or dichlorophenol indophenol with NADH as the electron donor. The diaphorase activity is located in the small subunit.
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PMID:The assimilatory nitrate reductase from the phototrophic bacterium, Rhodobacter capsulatus E1F1, is a flavoprotein. 930 29

We have synthesized a number of nitrobenzimidazoles containing nitro groups in the benzene ring and found that they acted as relatively efficient substrates for rat liver DT-diaphorase (EC 1.6.99.2), their reactivity exceeding reactivities of nitrofurans and nitrobenzenes. Nitrobenzimidazoles were competitive with NADPH inhibitors of DT-diaphorase in menadione reductase reactions, their inhibition constant being unchanged in the presence of dicumarol and being increased in the presence of 2',5'-ADP. These data indicate that the poor reactivity of nitrobenzimidazoles and other nitroaromatics in comparison to quinones could be determined by their binding in the adenosine-phosphate binding region of the NADPH-binding site, whereas quinones bind at the nicotinamide-binding pocket at the vicinity of FAD of DT-diaphorase. The reduction of 4,5,6-trinitrobenzimidazol-2-one by DT-diaphorase most probably involves reduction of 5-nitro group to 5-nitroso or 5-hydroxylamine derivative at the initial step. A certain parallelism existed between reactivities of nitrobenzimidazoles toward DT-diaphorase and their reactivities in single-electron reduction by Anabaena ferredoxin:NADP+ reductase (EC 1.18.1.2) and Saccharomyces cerevisiae flavocytochrome b2 (EC 1.1.2.3), the latter being determined by electronic factors. However, we suppose that the relatively high reactivity of polinitrobenzimidazoles toward DT-diaphorase was due not only to electronic effects, but also to a sterical crowding of nitrogroups by each other. The toxicity of nitrobenzimidazoles to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) with a moderate amount of DT-diaphorase (260 U/mg protein) is partly prevented by dicumarol. That points out to partial determination of nitrobenzimidazole cytotoxicity by their reduction by DT-diaphorase. Another important factor of nitrobenzimidazole toxicity to this cell line was oxidative stress, catalyzed by single-electron transferring enzymes.
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PMID:Nitrobenzimidazoles as substrates for DT-diaphorase and redox cycling compounds: their enzymatic reactions and cytotoxicity. 934 69

Human NAD(P)H:quinone acceptor oxidoreductase-2 (NQO2) has been prepared using an Escherichia coli expression method. NQO2 is thought to be an isoform of DT-diaphorase (EC 1.6.99.2) [also referred to as NAD(P)H:quinone acceptor oxidoreductase] because there is a 49% identity between their amino acid sequences. The present investigation has revealed that like DT-diaphorase, NQO2 is a dimer enzyme with one FAD prosthetic group per subunit. Interestingly, NQO2 uses dihydronicotinamide riboside (NRH) rather than NAD(P)H as an electron donor. It catalyzes a two-electron reduction of quinones and oxidation-reduction dyes. One-electron acceptors, such as potassium ferricyanide, cannot be reduced by NQO2. This enzyme also catalyzes a four-electron reduction, using methyl red as the electron acceptor. The NRH-methyl red reductase activity of NQO2 is 11 times the NADH-methyl red reductase activity of DT-diaphorase. In addition, through a four-electron reduction reaction, NQO2 can catalyze nitroreduction of cytotoxic compound CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. NQO2 is 3000 times more effective than DT-diaphorase in the reduction of CB 1954. Therefore, NQO2 is a NRH-dependent oxidoreductase which catalyzes two- and four-electron reduction reactions. NQO2 is resistant to typical inhibitors of DT-diaphorase, such as dicumarol, Cibacron blue, and phenindone. Flavones are inhibitors of NQO2. However, structural requirements of flavones for the inhibition of NQO2 are different from those for DT-diaphorase. The most potent flavone inhibitor tested so far is quercetin (3,5,7,3',4'-. 6pentahydroxyflavone). It has been found that quercetin is a competitive inhibitor with respect to NRH (Ki = 21 nM). NQO2 is 43 amino acids shorter than DT-diaphorase, and it has been suggested that the carboxyl terminus of DT-diaphorase plays a role in substrate binding (S. Chen et al., Protein Sci. 3, 51-57, 1994). In order to understand better the basis of catalytic differences between NQO2 and DT-diaphorase, a human NQO2 with 43 amino acids from the carboxyl terminus of human DT-diaphorase (i.e., hNQO2-hDT43) has been prepared. hNQO2-hDT43 still uses NRH as an electron donor. In addition, the chimeric enzyme is inhibited by quercetin but not dicumarol. These results suggest that additional region(s) in these enzymes is involved in differentiating NRH from NAD(P)H.
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PMID:Catalytic properties of NAD(P)H:quinone oxidoreductase-2 (NQO2), a dihydronicotinamide riboside dependent oxidoreductase. 936 28

The petH genes encoding ferredoxin:NADP+ reductase (FNR) from two Anabaena species (PCC 7119 and ATCC 29413) were cloned and overexpressed in E. coli. Several positively charged residues (Arg, Lys) have been implicated to be involved in ferredoxin binding and electron transfer by cross-linking, chemical modification and protection experiments, and crystallographic studies. The following substitutions were introduced by site-directed mutagenesis: R153Q, K209Q, K212Q, R214Q, K275N, K430Q and K431Q in Anabaena 29413 FNR, and R153E, K209E, K212E, R214E, K275E, R401E, K427E, and K431E in Anabaena 7119 FNR. Comparison of the diaphorase activities, the specific rates of ferredoxin dependent NADP(+)-photoreduction and cytochrome c reduction catalyzed by FNR showed that all these amino acid residues were required for efficient electron transfer between FNR and ferredoxin. Replacement of any one of these basic residues produced a much more pronounced effect on the cytochrome c reductase activity, where FNR, reduced by NADPH, acted as electron donor, than in the reduction of NADP+ by photosystem I via FNR. A mutation involving the replacement of positive charge by a neutral amide produced in all cases a smaller inhibitory effect on the activity than a charge reversal mutation. In addition, it has been found that R214 was necessary for stable integration of the non covalently bound FAD-cofactor.
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PMID:Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin:NADP+ reductase with ferredoxin probed by site directed mutagenesis. 951 8

In view of the ubiquitous role of the thioredoxin/thioredoxin reductase (TRX/TR) system in living cells, the interaction of Arabidopsis thaliana NADPH-thioredoxin reductase (EC 1.6.4.5) with quinones, an important class of redox cycling and alkylating xenobiotics, was studied. The steady-state reactions of A. thaliana TR with thioredoxin (TRX) and reaction product NADP+ inhibition patterns were in agreement with a proposed model of E. coli enzyme (B.W. Lennon, C.H. Williams, Jr., Biochemistry, vol. 35 (1996), pp. 4704-4712), that involved enzyme cycling between four- and two-electron reduced forms with FAD being reduced. Quinone reduction by TR proceeded via a mixed single- and two-electron transfer, the percentage of single-electron flux being equal to 12-16%. Bimolecular rate constants of quinone reduction (kcat/km) and reaction catalytic constants (kcat) increased upon an increase in quinone single-electron reduction potential. E(1)7. In several cases, the kcat of quinone reduction exceeded kcat of TRX reduction, suggesting that quinones intercepted electron flux from TR to TRX. Incubation of reduced TR with alkylating quinones resulted in a rapid loss of TRX-reductase activity, while quinone reduction rate was unchanged. In TRX-reductase and quinone reductase reactions of TR, NADP+ exhibited different inhibition patterns. These data point out that FAD and not the catalytic disulfide of TR is responsible for quinone reduction, and that quinones may oxidize FADH2 before it reduces catalytic disulfide. Most probably, quinones may oxidize the two-electron reduced form of TR, and the enzyme may cycle between two-electron reduced and oxidized forms in this reaction. The relatively high rate of quinone reduction by A. thaliana thioredoxin reductase accompanied by their redox cycling, confers pro-oxidant properties to this antioxidant enzyme. These factors make plant TR an attractive target for redox active and alkylating pesticide action.
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PMID:Interaction of quinones with Arabidopsis thaliana thioredoxin reductase. 954 49

One of the major mechanism of chemical protection against mutagenesis, carcinogenesis and other forms of toxicity is the induction of phase-II metabolizing enzymes such as UDP-glucuronosyl transferases, glutathione S-transferases and NAD(P)H quinone reductase, or inhibition of typical phase-I reactions. The use of selective inducers of conjugating enzymes or inhibitors of CYP- and FAD-dependent monooxygenases revealed the possibility of reducing the expression of certain forms of malignancy. However, the use of some anti-initiating entities devised to reduce tumor initiation, seems to receive invalidated justification. Indeed, considering the double edge-sword nature (activating or detoxifying) of drug metabolizing enzymes as well as the myriad of xenobiotics to which human is exposed, any attempt to modulate such catalysts by dietary components (including drugs) could lead to an increased cancer risk. Paradoxically, it has been recently proposed the use of metabolizing liver preparations, isolated from phase-II induced rodents, as a novel bioactivating model in the field of genetic toxicology. Exogenous microsomal (S9) fraction prepared from 2-(3)-tert-butyl-4-hydroxyanisole (BHA) (monofunctional post-oxidative inducer) treated mice are able to increase the DNA binding and genotoxic response of pre-mutagens. On the whole, the use of enzyme modulators in cancer chemoprevention, for their ability to simultaneously reduce or increase pre-carcinogen bioactivation, should be carefully reconsidered.
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PMID:The pitfall of detoxifying enzymes. 967 74

Recently a newly discovered pyridine nucleotide-disulfide oxidoreductase was reported to be essential for the degradation of epoxyalkanes by the Xanthobacter Py2 [Swaving, J., De Bont, J. A. M., Westphal, A. & De Kok, A. (1996) J. Bacteriol. 178, 6644-6646]. The disulfide oxidoreductase has now been purified from propene-grown Xanthobacter Py2. This enzyme (component II) is a NADPH-dependent FAD-containing homodimeric protein. The physiological substrate for this enzyme is unknown. The enzyme was active with the following dithiol substrates in decreasing order: 1,3-propanedithiol, reduced lipoamide and dithiothreitol, and inactive with glutathione and monothiols. In the reversed direction, only activity with 5,5'-dithiobis(2-nitrobenzoate) could be measured. Compared with other disulfide reductases it has a high activity with 5,5'-dithiobis(2-nitrobenzoate) and a low diaphorase and oxidase activity. Steady-state kinetic studies at pH 8.5 with 1,3-propanedithiol show that the enzyme operates by a ternary complex mechanism in the direction of NADP+ reduction. Anaerobic incubation of the enzyme with 1,3-propanedithiol resulted in slow reduction of the enzyme to yield the thiolate-FAD charge-transfer complex, the rate depending on the pH. At pH 7, where reduction was not detectable within 2 h, rapid mixing of NADP+ with the enzyme-propanedithiol mixture resulted in the formation of a complex between the reduced enzyme and NADP+ within the dead time of the instrument (5.6 ms). This is followed by slow formation of NADPH, concomitant with the appearance of the flavin C(4a)-thiol adduct, as judged from the spectral changes. This suggests that the rate-limiting step is the transfer of a hydride ion from the half-reduced enzyme to NADP+. Stopped-flow experiments involving reduction by NADPH show a biphasic behavior. The rapid formation (k(obs) = 40 s(-1)) of a transient intermediate with little absorption decrease at 460 nm and long wavelength absorption was followed by the slow formation (k(obs) = 4 s(-1)) of a species characterized as the thiolate-FAD charge-transfer complex with bound NADP+. Some formation of the FAD C(4a)-thiol adduct was also observed. Photoreduction in the presence of deazaflavin results in rapid bleaching at 450 nm, followed by the slow formation of a stable semiquinone. Full reduction could not be achieved, either by photoreduction or with NADPH, and was incomplete even with dithionite or NADPH in the presence of arsenite. The results indicate a low redox potential of the FAD and a slow rate of electron transfer from the pyridine nucleotide to the redox active disulfide and vice versa. From a sequence alignment with other disulfide reductases, it appears that the active site His-Glu diad is absent in this enzyme. The kinetic and spectral features described above will be discussed in this context.
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PMID:Purification and characterization of a flavoprotein involved in the degradation of epoxyalkanes by Xanthobacter Py2. 979 15

In mammals, two separate but homologous cytosolic quinone reductases have been identified: NAD(P)H:quinone oxidoreductase type 1 (QR1) (EC 1.6.99.2) and quinone reductase type 2 (QR2). Although QR1 and QR2 are nearly 50% identical in protein sequence, they display markedly different catalytic properties and substrate specificities. We report here two crystal structures of QR2: in its native form and bound to menadione (vitamin K(3)), a physiological substrate. Phases were obtained by molecular replacement, using our previously determined rat QR1 structure as the search model. QR2 shares the overall fold of the major catalytic domain of QR1, but lacks the smaller C-terminal domain. The FAD binding sites of QR1 and QR2 are very similar, but their hydride donor binding sites are considerably different. Unexpectedly, we found that QR2 contains a specific metal binding site, which is not present in QR1. Two histidine nitrogens, one cysteine thiol, and a main chain carbonyl group are involved in metal coordination. The metal binding site is solvent-accessible, and is separated from the FAD cofactor by a distance of about 13 A.
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PMID:Crystal structure of human quinone reductase type 2, a metalloflavoprotein. 1043 94


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