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 salivary glands of the hematophagous insect, Rhodnius prolixus, contain a nitrosylhemeprotein that dissociates its ligand, NO, to the host tissues while the insect is searching for a blood meal. We now report a salivary nitric oxide synthase activity in this insect. The activity is dependent on NADPH, FAD, tetrahydrobiopterin, calmodulin, Ca2+, and converts arginine to citrulline while producing vasorelaxing activity. Molecular sieving indicates a molecular weight of 185 kDa, coeluting with a diaphorase activity. Results indicate similarity of this insect activity to the vertebrate constitutive NO synthase, suggesting NO synthesis is an evolutionary old biological pathway.
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PMID:Nitric oxide synthase activity from a hematophagous insect salivary gland. 768 81

Genetic analysis of a patient with the variant cytochrome b-245-positive form of chronic granulomatous disease revealed a missense mutation resulting in a Arg54-->Ser substitution in the gp91phox subunit of cytochrome b-245. As a consequence, although no O2- is made, NADPH oxidase-associated FAD accepts electrons from NADPH in the cell-free activation system and becomes reduced. The reduced flavin exhibits normal levels of iodonitrotetrazolium violet diaphorase activity, and the patient's neutrophils exhibit high levels of intracellular oxidant production and show a low level of NBT staining in the NBT slide test. Thus, this mutation appears to render the heme center of NADPH oxidase present but nonfunctional, while leaving the flavin center fully functional.
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PMID:A variant X-linked chronic granulomatous disease patient (X91+) with partially functional cytochrome b. 771 25

NADH peroxidase is a flavoenzyme having a single redox-active thiol, Cys42, that cycles between sulfenate and thiol forms in the NADH-dependent reduction of hydrogen peroxide. NADH peroxidase catalyzes the NADH-dependent reduction of quinones with turnover numbers between 1.2 and 3.9 s-1, per mole of FAD, at pH 7.5. The bimolecular rate constants for quinone reduction, V/K, ranged from 4.3 x 10(3) to 6.0 x 10(5) M-1 s-1 for 14 quinones whose redox potentials varied between -0.41 and 0.09 V. The logarithms of the V/K values for these quinones are hyperbolically dependent on their single-electron reduction potentials (E7(1). One-electron reduction of benzoquinone accounts for about 50% of the total electron transfer catalyzed by NADH peroxidase at pH 7, with the remainder of the reduction being catalyzed by a two-electron (hydride) transfer. Cys42 can be irreversibly oxidized to the sulfonate by hydrogen peroxide, with inactivation of the peroxidatic activity of the enzyme. The residual quinone reductase activity of NADH peroxidase which has undergone oxidative inactivation of the active site Cys42 indicates that this residue is not involved in the reduction of the quinones. Product inhibition studies suggest the possibility of overlap of the pyridine nucleotide and quinone binding sites in the reduced enzyme at low pH values. The pH dependence of the maximum velocity of naphthoquinone reduction shows that deprotonation of an enzymic group, exhibiting a pK value of ca. 6.2, decreases the maximal velocity. Primary deuterium kinetic isotope effects on V and V/K for quinone-dependent NADH oxidation increase upon protonation of a group, exhibiting a pK value of 6.4.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Quinone reductase reaction catalyzed by Streptococcus faecalis NADH peroxidase. 775 94

The respiratory chain of marine and moderately halophilic bacteria requires Na+ for maximum activity, and the site of Na(+)-dependent activation is located in the NADH-quinone reductase segment. The Na(+)-dependent NADH-quinone reductase purified from marine bacterium Vibrio alginolyticus is composed of three subunits, alpha, beta, and gamma, with apparent M(r) of 52, 46, and 32 kDa, respectively. The FAD-containing beta-subunit reacts with NADH and reduces ubiquinone-1 (Q-1) by a one-electron transfer pathway to produce ubisemiquinones. In the presence of the FMN-containing alpha-subunit and the gamma-subunit, Q-1 is converted to ubiquinol-1 without the accumulation of free radicals. The reaction catalyzed by the alpha-subunit is strictly dependent on Na+ and is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), which is tightly coupled to the electrogenic extrusion of Na+. A similar type of Na(+)-translocating NADH-quinone reductase is widely distributed among marine and moderately halophilic bacteria. The respiratory chain of V. alginolyticus contains another NADH-quinone reductase which is Na+ independent and has no energy-transducing capacity. These two types of NADH-quinone reductase are quite different with respect to their mode of quinone reduction and their sensitivity toward NADH preincubation.
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PMID:Na(+)-translocating NADH-quinone reductase of marine and halophilic bacteria. 822 20

The carboxyl-terminal region of plant ferredoxin-NADP+ reductases is formed by an invariant alpha-helix/loop/beta-strand, culminating in a conserved tyrosine that displays extensive interaction with the prosthetic group FAD. We have investigated the potential role of the terminal region in reductase function, by introducing mutations and deletions on pea ferredoxin-NADP+ reductase overexpressed in Escherichia coli. Replacement of the terminal tyrosine by tryptophan, phenylalanine, serine, and glycine resulted in a 2.2-, 2.0-, 22-, and 302-fold reduction, respectively, in kcat for the diaphorase reaction, whereas elimination of the tyrosine caused a 846-fold decrease in kcat. Km values were largely unaffected by the substitutions. Similar results were obtained when the mutants were assayed for cytochrome c reduction, indicating that aromaticity is the most important factor to the function of the tyrosine in catalysis. The presence of the phenol ring at the carboxyl-terminal position of wild-type reductase is important, but not an absolute requirement for enzyme function or FAD assembly. Deletion of the alpha-helix/beta-strand region prevented reductase proper folding in the bacterial host, while shortening of the terminal region by splicing 3 amino acids at the beginning of the alpha-helix produced a moderately soluble reductase, devoid of FAD and enzymatic activity.
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PMID:Probing the role of the carboxyl-terminal region of ferredoxin-NADP+ reductase by site-directed mutagenesis and deletion analysis. 836 77

Nitrate reductase from the yeast Candida nitratophila was found to contain one molecule of cytochrome b557 and one atom of molybdenum per subunit. FAD/haem-dependent diaphorase activity (haem domain) was associated with a 40 kDa tryptic fragment of the subunit. The 50 amino-terminal residues of this fragment were determined, and the sequence did not show significant similarity to deduced sequences of other nitrate reductases previously published. Increasing ionic strength in vitro had a stimulatory effect on enzymic activity via stimulation of the molybdenum-dependent terminal nitrate-reducing activity. Stimulation of activity by exogenous protein (bovine serum albumin or casein) also appeared to be an ionic effect. Stimulation of catalytic activity by phosphate was a separate effect.
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PMID:Further characterization of the assimilatory nitrate reductase from the yeast Candida nitratophila. 847 56

To investigate the functional role of the cysteine residues present in the spinach ferredoxin-NADP+ oxidoreductase, we individually replaced each of the five cysteine residues with serine using site-directed mutagenesis. All of the mutant reductases were correctly assembled in Escherichia coli except for the C42S mutant protein. C114S and C137S mutant enzymes apparently showed structural and kinetic properties very similar to those of the wild-type reductase. However, C272S and C132S mutations yielded enzymes with a decreased catalytic activity in the ferredoxin-dependent reaction (14 and 31% of the wild type, respectively). Whereas the C132S was fully competent in the diaphorase reaction, the C272S mutant flavoprotein showed a 35-fold reduction in catalytic efficiency with respect to the wild-type enzyme (0.4 versus 14.28 microM-1 s-1) due to a substantial decrease of kcat. NADP+ binding by the C272S mutant enzyme was apparently quantitatively the same (Kd = 37 microM) but qualitatively different, as shown by the differential spectrum. Stopped-flow experiments showed that the enzyme-FAD reduction rate was considerably decreased in the C272S mutant reductase, along with a much lower yield of the charge-transfer transient species. It is inferred from these data that the charge transfer (FAD-NADPH) between the reductase and NADPH is required for hydride transfer from the pyridine nucleotide to flavin to occur with a rate compatible with catalysis.
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PMID:The role of cysteine residues of spinach ferredoxin-NADP+ reductase As assessed by site-directed mutagenesis. 851 83

Incubation of either Chlorella nitrate reductase or the recombinant flavin domain of spinach nitrate reductase with reagents specific for modification of cysteine residues, such as N-ethylmaleimide, resulted in a time-dependent inactivation of NADH:ferricyanide reductase activity which could be prevented by incubation in the presence of NADH. At 25 degrees C and employing a fixed enzyme:modifier ratio, the rate of inactivation for both the Chlorella and spinach enzymes followed the order p-chloromercuribenzoate > methyl methanethiosulfonate > 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid > N-ethylmaleimide. For the spinach flavin domain, inactivation by methyl methanethiosulfonate or p-chloromercuribenzoate was found to be concentration independent suggesting the absence of nonspecific modifications. Initial rate studies of the methyl methanethiosulfonate-modified flavin domain indicated a reduction in NADH:ferricyanide activity (Vmax) from 85 to 44 micromol NADH consumed/min/nmol FAD and an increase in the Km for NADH from 12 to 35 microM when compared to the native enzyme, confirming a role for cysteine residue(s) in maintaining diaphorase activity. Site-directed mutagenesis of the four individual cysteines (residues 17, 54, 62, and 240) in the recombinant spinach flavin domain resulted in mutant proteins with visible and CD spectra very similar to those of the wild-type domain. Initial rate studies indicated that only substitutions of serine for cysteine 240 decreased diaphorase activity with maximal NADH:ferricyanide activity for the C240S mutant corresponding to 51 micromol NADH consumed/min/nmol FAD with a Km for NADH of 14 microM. Mutation of C240 to Ala or Gly resulted in greater loss of activity. The thermal stability of the four serine mutants was slightly decreased compared to the wild-type domain with the C62S mutant exhibiting the greatest instability. In contrast to the effects on diaphorase activity, square wave voltammetric studies indicated changes in the oxidation-reduction midpoint potential for the FAD/FADH2 couple in the C54S (E0'= -197 mV), C62S (E0' = -226 mV), and C240S (E0' = -219 mV) mutants compared to the wild-type domain (E0' = -268 mV). These results indicate that of the four cysteine residues in the spinach nitrate reductase flavin domain, only C240 plays a role in maintaining diaphorase activity, while C54 has the greatest influence on flavin redox potential and that no correlation between changes in catalytic activity and flavin redox potential was observed.
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PMID:Thiol modification and site directed mutagenesis of the flavin domain of spinach NADH:nitrate reductase. 866 Jun 90

Cytosolic NADPH-dependent ubiquinone reductase (NADPH-UQ reductase) accounted for about 68% of the total ubiquinone (UQ) reductase activity in rat liver homogenate [Takahashi, T. et al. (1995) Biochem. J. 309, 883-890]. We investigated the effects of various factors on this enzyme activity in rat liver cytosol with the aim of elucidating its physiological roles. The NADPH-UQ reductase in rat liver cytosol catalyzed the reduction of UQ to UQH2 with concomitant oxidation of equimolar NADPH. The optimal pH was around 7.4, and the optimal temperatures were about 28 degrees C for NADH and about 37 degrees C for NADPH. NADH, deamino NADH, and deamino NADPH were much less active hydrogen donors than NADPH, whereas reduced nicotinamide mononucleotide, ascorbate, erythorbate, reduced glutathione, and cysteine were inactive. As the hydrogen acceptor, UQ-9 had the highest Vmax/Km among the long-chain UQ homologues tested. FAD and FMN stimulated the activity. Anionic detergents, Mg2+ and Sr2+ also enhanced the activity. Rotenone, malonic acid, antimycin A, and KCN, which inhibit mitochondrial and microsomal electron transfer enzymes, superoxide dismutase, and acetylated cytochrome c had no effect on the NADPH-UQ reductase activity. These results indicated that the NADPH-UQ reductase in rat liver cytosol is a flavoprotein that reduces UQ-10 by a two-electron reduction mechanism and is distinguishable from known microsomal and mitochondrial enzymes, as well as DT-diaphorase [EC 1.6.99.2].
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PMID:Characterization of NADPH-dependent ubiquinone reductase activity in rat liver cytosol: effect of various factors on ubiquinone-reducing activity and discrimination from other quinone reductases. 888 15

A mammalian cytosolic FAD-dependent enzyme that catalyzes the reduction of quinones by N-ribosyl- and N-alkyldihydronicotinamides, but not by NADH, NADPH, or NMNH (reduced nicotinamide mononucleotide), was isolated from bovine kidney more than 30 years ago [S. Liao, J. T. Dulaney and H. G. Williams-Ashman (1962) J. Biol. Chem. 237, 2981-2987]. This enzyme is designated here as quinone reductase type 2 (QR2). Bovine QR2 is a homodimer that migrates on SDS/PAGE at approximately 22 kDa. Three tryptic peptides of bovine QR2 (representing 39 amino acids) showed 43% identity to human NAD(P)H:quinone reductase (DT-diaphorase; EC 1.6.99.2), here designated QR1 and 82% identity to a related human cDNA clone [called hNQO2 by A. K. Jaiswal, P. Burnett, M. Adesnik and O. W. McBride (1990) Biochemistry 29, 1899-1906], and designated here as hQR2. The protein encoded by the latter cDNA did not show QR activity when tested with conventional nicotinamide nucleotides. The unexpected high homology between the old flavoenzyme and hQR2 prompted us to clone and overexpress hQR2. The properties of hQR2 were identical to those of the flavoenzyme described by S. Liao and H. G. Williams-Ashman, thus establishing their genetic identity. Recombinant human QR2: (i) reacts with N-ribosyl- and N-alkyldihydronicotinamides, but not with NADH, NADPH, or NMNH; (ii) is very weakly inhibited by dicumarol or Cibacron blue; (iii) is very potently inhibited by benzo[a]pyrene. The x-ray crystal structure of rat QR1 shows that the 43 amino acid C-terminal tail of QR1 provides the binding site for the hydrophilic portions of NADH and NADPH. In the absence of this binding site in QR2, the enzyme retains the essential catalytic machinery, including affinity for FAD, but cannot bind phosphorylated hydride donors.
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PMID:Unexpected genetic and structural relationships of a long-forgotten flavoenzyme to NAD(P)H:quinone reductase (DT-diaphorase) 905 Aug 36


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