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 cytochrome b subunit of the bc1 complexes contains two cytochrome components (bL and bH) and is the locus of both a quinol-oxidizing site (Qo or Qz) and a quinone-reducing site (Qi or Qc). Sequence alignments of this subunit from over 20 eukaryotic and prokaryotic species have revealed a remarkable degree of conservation, including approximately 20 totally conserved residues. In this paper, site-directed mutagenesis has been used to examine the structural or functional roles of 5 of these highly conserved residues, Gly48, Gln58, Ser102, Phe104, and Pro202, all predicted to be within transmembrane alpha-helical segments. The mutants were made in the bc1 complex of Rhodobacter sphaeroides, a photosynthetic bacterium. The ability to use spectroscopic, electrochemical, and flash-induced kinetic methods allows the mutants to be analyzed for influences both on cytochrome spectra and thermodynamic properties and on the kinetics of specific electron transfer reactions. The results show that none of the 5 residues is absolutely essential. Substitution of aspartate or valine for Gly48 results in the loss of photosynthetic growth. The G48V mutant assembles a bc1 complex, but with modified cytochromes bH and bL, and a dysfunctional quinone reductase (Qc) site; an alanine is tolerated at this position. Possibly, a small residue is important here for heme packing. Gln58 and Ser102 are the only highly conserved polar residues predicted to be within the transmembrane spans, apart from the histidines which are heme axial ligands. Neither Gln58 nor Ser102 is essential for assembly or function of the bc1 complex, although substitution of other amino acids in these positions does cause subtle, but measurable changes. Phe104 lies midway between the axial ligands to cytochromes bL and bH and can be modeled to project in the space separating the two hemes. Replacement of this highly conserved aromatic residue by isoleucine has no measurable influence on the rate of electron transfer through the cytochrome b chain containing the two hemes. Finally, Pro202 is a totally conserved proline which is in the middle of transmembrane helix D, in between the 2 histidines which provide ligands to the hemes. No major inhibition of electron transfer resulted from replacing this proline by a leucine, although subtle changes in spectra of the b cytochromes and their electrochemical properties were noted.
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PMID:Examination of the functional roles of 5 highly conserved residues in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides. 131 21

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

A prokaryotic expression plasmid, pKK-DT2, containing the cDNA of rat liver NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.99.2; DT-diaphorase) was constructed and used to transform Escherichia coli strain JM109. The rat liver quinone reductase was expressed in strain in JM109 and was inducible with isopropyl beta-D-thiogalactopyranoside (IPTG). The expressed rat protein was purified by affinity chromatography and had kinetic and physical properties identical with the protein purified from rat liver in that it could utilize either NADH or NADPH as the electron donor and its activity was inhibited by dicoumarol. In addition, we have generated four mutants, Arg-177----His (R177H), Arg-177----Ala (R177A), Arg-177----Cys (R177C) and Arg-177----Leu (R177L), using this expression system. Several of the mutants behaved anomalously on SDS/PAGE, but all of the mutant proteins had the expected M(r) as determined by electrospray m.s. These results and those obtained from enzyme kinetic analysis, u.v./visible absorption spectral analysis, and flavin and tryptophan fluorescence analysis of the wild-type enzyme and four mutants indicated that mutations at Arg-177 changed the conformation of the enzyme, resulting in a decrease in enzyme activity. Replacing Arg-177 with leucine altered the protein conformation and decreased FAD incorporation.
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PMID:Expression of rat liver NAD(P)H:quinone-acceptor oxidoreductase in Escherichia coli and mutagenesis in vitro at Arg-177. 162 1

A full-length cDNA clone, pKK-DTD4, complementary to rat liver cytosolic DT-diaphorase [NAD(P)H:quinone oxidoreductase (EC 1.6.99.2)] mRNA was expressed in Escherichia coli. The pKK-DTD4 cDNA was obtained by extending the 5'-end sequence of a rat liver DT-diaphorase cDNA clone, pDTD55, to include an ATG initiation codon and the NH2-terminal codons using polymerase chain reaction (PCR). Restriction sites for EcoRI and HindIII were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by the PCR reaction. The resulting full-length cDNA was inserted into an expression vector, pKK2.7, at the EcoRI and HindIII restriction sites. E. coli strain AB1899 was transformed with the constructed expression plasmid, and DT-diaphorase was expressed under the control of the tac promotor. The expressed DT-diaphorase exhibited high activity of menadione reduction and was inhibited by dicumarol at a concentration of 10(-5)M. After purification by Cibacron Blue affinity chromatography, the expressed enzyme migrated as a single band on 12.5% sodium dodecyl sulfate-polyacrylamide gel with a molecular weight equivalent to that of the purified rat liver cytosolic DT-diaphorase. The purified expressed protein was recognized by polyclonal antibodies against rat liver DT-diaphorase on immunoblot analysis. It utilized either NADPH or NADH as electron donor at equal efficiency and displayed high activities in reduction of menadione, 1,4-benzoquinone, and 2,6-dichlorophenolindophenol which are typical substrates for DT-diaphorase. The expressed DT-diaphorase exhibited a typical flavoprotein spectrum with absorption peaks at 380 and 452 nm. Flavin content determination showed that it contained 2 mol of FAD per mole of the enzyme. Edman protein sequencing of the first 20 amino acid residues at the NH2 terminus of the expressed protein indicated that the expressed DT-diaphorase is not blocked at the NH2 terminus and has an alanine as the first amino acid. The remaining 19 amino acid residues at the NH2 terminus were identical with those of the DT-diaphorase purified from rat liver cytosol.
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PMID:Expression of mammalian DT-diaphorase in Escherichia coli: purification and characterization of the expressed protein. 170 98

A new method for the measurement of urinary dipeptidase activity is described. The action of dipeptidase on L-Ala-L-Ala results in production of an L-alanine, and this amino acid is simultaneously determined by an L-alanine dehydrogenase-diaphorase system. As urinary substances do not affect this reaction, the measurement can be accomplished without prior dialysis. The mean value +/- S.D. for normals was found to be 12.0 +/- 4.4 IU/g of creatinine. Elevated values were found in chronic nephritis (55.9 +/- 35.0 IU/g of creatinine, P less than 0.001 vs. normal), acute nephritis (46.6 +/- 29.9 IU/g of creatinine, P less than 0.001), and nephrotic syndrome (43.3 +/- 36.5 IU/g of creatinine, P less than 0.001). The dipeptidase activity thus measured showed a significant correlation with dipeptidase activity against L-Leu-L-Leu as substrate. On disc polyacrylamide gel electrophoresis, the urinary dipeptidase of a patient with chronic nephritis appeared as one band with similar mobility to human kidney dipeptidase F. Urinary dipeptidase in a patient with chronic nephritis was identical to human kidney dipeptidase on double immunodiffusion analysis.
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PMID:A fluorometric method for dipeptidase activity measurement in urine, using L-alanyl-L-alanine as substrate. 643 29

Enhanced formation of nitric oxide (NO) by both the constitutive and the inducible isoforms of NO synthase (NOS) has been implicated in the pathophysiology of a variety of diseases, including circulatory shock. Non-isoform-selective inhibition of NO formation, however, may lead to side effects by inhibiting the constitutive isoform of NOS and, thus, the various physiological actions of NO. S-Methylisothiourea sulfate (SMT) is at least 10- to 30-fold more potent as an inhibitor of inducible NOS (iNOS) in immunostimulated cultured macrophages (EC50, 6 microM) and vascular smooth muscle cells (EC50, 2 microM) than NG-methyl-L-arginine (MeArg) or any other NOS inhibitor yet known. The effect of SMT on iNOS activity can be reversed by excess L-arginine in a concentration-dependent manner. SMT (up to 1 mM) does not inhibit the activity of xanthine oxidase, diaphorase, lactate dehydrogenase, monoamine oxidase, catalase, cytochrome P450, or superoxide dismutase. SMT is equipotent with MeArg in inhibiting the endothelial, constitutive isoform of NOS in vitro and causes increases in blood pressure similar to those produced by MeArg in normal rats. SMT, however, dose-dependently reverses (0.01-3 mg/kg) the hypotension and the vascular hyporeactivity to vasoconstrictor agents caused by endotoxin [bacterial lipopolysaccharide (LPS), 10 mg/kg, i.v.] in anesthetized rats. Moreover, therapeutic administration of SMT (5 mg/kg, i.p., given 2 hr after LPS, 10 mg/kg, i.p.) attenuates the rises in plasma alanine and aspartate aminotransferases, bilirubin, and creatinine and also prevents hypocalcaemia when measured 6 hr after administration of LPS. SMT (1 mg/kg, i.p.) improves 24-hr survival of mice treated with a high dose of LPS (60 mg/kg, i.p.). Thus, SMT is a potent and selective inhibitor of iNOS and exerts beneficial effects in rodent models of septic shock. SMT, therefore, may have considerable value in the therapy of circulatory shock of various etiologies and other pathophysiological conditions associated with induction of iNOS.
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PMID:Beneficial effects and improved survival in rodent models of septic shock with S-methylisothiourea sulfate, a potent and selective inhibitor of inducible nitric oxide synthase. 752 23

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

NAD(P)H:quinone oxidoreductase (NQOR; EC 1.6.99.2) is a homodimeric enzyme which catalyzes the reduction of quinones, azo dyes, and other electron acceptors by NADPH or NADH. To pursue subunit functional studies, we expressed a wild-type/mutant heterodimer of NQOR in Escherichia coli. The wild-type subunit of the heterodimer was tagged with polyhistidine and the other subunit contained a His-194-->Ala mutation (H194A), a change known to dramatically increase the Km for NADPH. This approach enabled us to efficiently purify the heterodimer (H194A/HNQOR) from the homodimers by stepwise elution with imidazole from a nickel nitrilotriacetate column under nondenaturing conditions. The composition of the purified heterodimer was confirmed by SDS and nondenaturing polyacrylamide gel electrophoresis and immunoblot analysis. The enzyme kinetics of the purified heterodimer were studied with two two-electron acceptors, 2,6-dichloroindophenol and menadione, and a four-electron acceptor, methyl red, as the substrates. With two-electron acceptors, the Km(NADPH) and Km(NADH) values of the heterodimer H194A/HNQOR were virtually identical to those of the wild-type homodimer, but the kcat-(NADPH) and kcat(NADH) values were only about 50% those of the wild-type homodimer. With the four-electron acceptor, the Km and kcat values of H194A/HNQOR for NADPH and NADH were similar to those of the low-efficiency mutant homodimer. These results suggest that the subunits of NQOR function independently with two-electron acceptors, but dependently with a four-electron acceptor. This heterodimer approach may have general applications for studying the functional and structural relationships of subunits in dimeric or oligomeric proteins.
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PMID:Subunit functional studies of NAD(P)H:quinone oxidoreductase with a heterodimer approach. 786 30

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

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


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