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)

A dihydrodipicolinate reductase containing flavin was purified from sporulating Bacillus subtilis PCI 219. The purified enzyme appeared homogeneous by dise gel electrophoresis. Its molecular weight was estimated as 74,000 by gel filtration on Sephadex G-200, and as 18,500 by electrophoresis on sodium dodecylsulfate polyacrylamid gel. These results suggest that the enzyme is composed of four subunits. The prosthetic group was identified as FMN, and one mole of the enzyme contained two moles of FMN. Both NADPH and NADH acted as coenzyme, though NADH was less effective. The enzyme also exhibited diaphorase activity. The pH optimum was 6.1. The enzyme was inhibited by dipicolinate but not by lysine or alpha, epsilon-diaminopimelate.
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PMID:A new flavin enzyme catalyzing the reduction of dihydrodipicolinate in sporulating Bacillus subtilis I. Purification and properties. 23 91

Ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 is chemically modified by pyridoxal 5'-phosphate. The incorporation of 2 +/- 0.3 mol pyridoxal 5'-phosphate/mol ferredoxin-NADP+ reductase inhibited NADPH-cytochrome c reductase activity by up to 95% while 55% of diaphorase activity still remained. Considerable protection against inactivation was afforded by ferredoxin. Chymotryptic cleavage of the modified enzyme was performed, the peptides were separated by high performance liquid chromatography, and the peptides containing pyridoxamine 5'-phosphate were identified by their fluorescence and by their absorbance at 325 nm. Three major labelled peptides were found. Their sequences were comprised of residues 46-54, 231-235 and 289-295. Lys-53 and -294 were the residues which presented the highest degree of modification and seem to be involved in the ferredoxin binding site of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119.
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PMID:Lysine residues on ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 involved in substrate binding. 154 17

We previously reported the expression of a full-length cDNA complementary to a rat liver NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) mRNA in Escherichia coli (Q. Ma, R. Wang, C. S. Yang, and A. Y. H. Lu, 1990, Arch. Biochem. Biophys. 283, 311-317). Since cysteine residues have been suggested to be important for the catalysis of flavoproteins and a lysine residue at position 76 in NAD(P)H:quinone oxidoreductase has been proposed to be involved in electron transfer of the enzyme, we investigated the roles of lysine 76 and cysteine 179 of this enzyme in catalysis by site-directed mutagenesis. Mutant cDNA clones replacing lysine 76 with valine (K76V) and cysteine 179 with alanine (C179A) were generated by a procedure based on the polymerase chain reaction. The mutant enzymes were expressed in E. coli. The cytosolic activities of the K76V and C179A mutants were 50 and 25% of that of the wild type (DTD), due to lower levels of the mutant proteins as shown by immunoblot analysis. The mutant proteins were purified to apparent homogeneity. The purified K76V and C179A mutant enzymes maintained full activities of 2,6-dichlorophenolindophenol (DCIP) reduction compared with that of the wild type. The mutant enzymes exhibited kinetic parameters for DCIP, NADH, and NADPH similar to those of DTD except that, with K76V, the Km for NADPH was doubled. Both mutant proteins contained two molecules of FAD per enzyme molecule. Dicumarol inhibited K76V and C179A mutant activities to greater than 90% at a concentration of 10(-7) M. Heat stability studies showed that C179A was much more sensitive to inactivation at 37 degrees C than both the wild-type and K76V enzymes. It is concluded from this study that lysine 76 and cysteine 179 are not essential in catalysis and in the binding of FAD, DCIP, and dicumarol. However, lysine residue 76 appears to play a role in NADPH binding and cysteine residue 179 is important in maintaining the stability of the enzyme.
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PMID:Site-directed mutagenesis of rat liver NAD(P)H: quinone oxidoreductase: roles of lysine 76 and cysteine 179. 156 99

The Namaqua is an indigenous fat-tailed African breed of sheep which has remained relatively isolated and which at one time dwindled to near extinction. Frequency data are given for blood group antigens, red cell glutathione and potassium types, for electrophoretic variants of red cell haemoglobin, 'X' protein, nucleoside phosphorylase, NADH-diaphorase, lysine and carbonic anhydrase and of plasma esterase, transferrin and albumin. Of particular interest was the occurrence of the i blood group, a bimodal distribution in red cell glutathione concentrations and red cell potassium concentrations of around 57 mmol/l cells, i.e. neither typically LK nor HK type.
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PMID:Blood groups and biochemical polymorphism in the Namaqua sheep breed. 261 Apr 3

The amino terminal blocked peptide of rat liver NAD(P)H:quinone reductase (DT-diaphorase) was determined by amino acid sequence analysis and by mass spectrometry. The mature protein is composed of 273 amino acids and contains an acetylated amino terminus, which was not identified by previous cDNA analysis. The enzyme was inactivated by p-nitrobenzenesulfonyl fluoride (NBSF) or 2,4,6-trinitrobenzenesulfonate (TNBS) with pseudo-first-order kinetics. These studies suggest that essential tyrosine and lysine may be present in the active site of this enzyme. The NBSF inhibition was protected by 1-naphthol and 1-naphthylamine, but not by NAD+. However, TNBS inhibition was not prevented by the naphthalene derivatives or NAD+. Specific peptides labeled with NBSF or TNBS were isolated by high-performance liquid chromatography and were sequenced. These analyses revealed that the NBSF-labeled tyrosine resides in a predominantly hydrophobic region and TNBS-labeled lysine in a predominantly hydrophilic region.
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PMID:Structure-function relationship of NAD(P)H:quinone reductase: characterization of NH2-terminal blocking group and essential tyrosine and lysine residues. 314 6

The triazine dyes, Cibacron blue F3GA and Procion red HE3B inhibited diaphorase activity of ferredoxin-NADP+ reductase, in a competitive manner with respect to NADPH. The Ki values were 1.5 and 0.2 microM, respectively. Binding of the dyes to the flavoprotein, as measured by difference spectroscopy, indicated an apparent stoichiometry of 1 mol dye/mol reductase and was prevented by NADP+ or high ionic strength. Chemical modification of a lysine residue and a carboxyl group at the NADP(H) binding site of the enzyme prevented complex formation with Procion red. Procion red showed a higher affinity for ferredoxin-NADP+ reductase than Cibacron blue. The Kd values were 1.9 and 5 microM, respectively. Once covalently linked to a Sepharose matrix, the triazine compounds specifically bind the flavoprotein. The interaction is partially electrostatic and partially hydrophobic. The enzyme can be eluted by high concentrations of salt or low concentrations of the corresponding coenzyme. The use of this affinity column allows the rapid purification of ferredoxin-NADP+ oxidoreductase from spinach leaves with good yields.
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PMID:Interaction of ferredoxin-NADP+ oxidoreductase with triazine dyes. A rapid purification method by affinity chromatography. 682 90

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

NAD(P)H: quinone-acceptor oxidoreductase (EC 1.6.99.2), also referred to as DT-diaphorase, is a flavoprotein that catalyzes the two-electron reduction of quinones and quinonoid compounds to hydroquinones, using either NADH or NADPH as the electron donor. Using an Escherichia coli expression system developed previously, we prepared three mutants of the rat liver quinone reductase. These mutants are Lys-113-His (K113H), Lys-113-Asp (K113D), and Lys-113-Ala (K113A). While the mutant K113H was readily purified using the same procedure as for the purification of the wild-type quinone reductase and found to have an activity similar to that of the wild-type enzyme, K113D and K113A were purified only in very small quantities, mainly in the form of apoprotein, and had very low activities. The results suggest that a positively charged amino acid at this position is important for the binding of the flavin adenine dinucleotide (FAD) prosthetic group. Flavin spectral studies of 6-mercapto-FAD-reconstituted mutants revealed that mutation at Lys-113 affects the protein environment around position-6 of the isoalloxazine ring.
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PMID:A site-directed mutagenesis study at Lys-113 of NAD(P)H:quinone-acceptor oxidoreductase: an involvement of Lys-113 in the binding of the flavin adenine dinucleotide prosthetic group. 763 39

A mutant of spinach ferredoxin-NADP+ reductase, in which Lys-88 has been changed to glutamine, has been obtained by site-directed mutagenesis. The mutant enzyme was fully active as a diaphorase, but partially impaired in ferredoxin-dependent cytochrome c reductase activity. By steady-state kinetics, the Km for ferredoxin of the K88Q enzyme was found to have increased 10-fold, whereas the kcat was unaffected by the amino acid replacement. The interaction between oxidized ferredoxin and the enzyme forms was also studied by spectrofluorimetric titration: Kd values of 110 and 10 nM were determined for the mutant and wild-type proteins, respectively. These data point out the importance of a positive charge at position 88 of the reductase for the interaction with ferredoxin, confirming previous cross-linking studies.
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PMID:Involvement of lysine-88 of spinach ferredoxin-NADP+ reductase in the interaction with ferredoxin. 817 9

Escherichia coli fumarate reductase (FRD) is a four-subunit enzyme that catalyzes the terminal step in anaerobic respiration to fumarate. The hydrophobic FrdC and FrdD subunits anchor the FrdA and FrdB catalytic subunits to the inner surface of the cytoplasmic membrane and are required for the enzyme to interact with quinones. Thirty-five single-site mutations were constructed in the FrdC and FrdD polypeptides by site-directed mutagenesis. Each mutant enzyme was characterized for its ability to catalyze quinone oxidation and reduction and to support growth of E. coli DW35 (delta frdABCD sdhC::kan) under selective conditions requiring functional enzyme. Replacement of FrdCE29 with Asp, Leu, Lys, or Phe had a deleterious effect both on quinol oxidase and quinone reductase activities. Substitution of FrdCH82 with Arg, Leu, Tyr, or Glu also decreased menaquinol oxidase activity, but had variable effects on the reverse reaction, the reduction of ubiquinone. Data are presented to support the hypothesis that the positive charge at FrdCH82 is required for stabilization of the quinone radical intermediate and the negative charge at FrdCE29 for deprotonation of menaquinol. Other critical amino acids identified in FrdC included Ala-32, Phe-38, Trp-86, Phe-87, and in FrdD residues Phe-57, Gln-59, Ser-60, and His-80. The established roles of such residues in the QA and QB sites of the photosynthetic reaction center would suggest a similar type of structure operative in the FRD complex. In such a model, Glu-29, Ala-32, His-82, Trp-86 of FrdC and His-80 of FrdD are considered participants in a QB-type site, and FrdD Phe-57, Gln-59, and Ser-60 components in an apolar QA-type site.
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PMID:Escherichia coli fumarate reductase frdC and frdD mutants. Identification of amino acid residues involved in catalytic activity with quinones. 841 59


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