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:1.8.1.4 (diaphorase)
2,754 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

The lipoic acids of the alpha-ketoglutarate dehydrogenase multienzyme complex from Escherichia coli have been modified with two fluorescent probes, N-(1-pyrenyl)-maleimide and 5-[[[(iodoacetyl)amino]ethyl]amino]-naphthylene-1-sulfonic acid. Time-resolved fluorescence polarization of partially labeled complexes (18-77% inhibition of enzyme activity) reveals a complex depolarization process: one component of the anisotropy is characterized by a rotational correlation time much longer than the time scale of the measurements (less than or equal to 400 ns), reflecting the overall rotation of the complex, while a second component of the anisotropy decays with a rotational correlation time of 320 (+/- 50) ns. This decay is essentially independent of viscosity and is consistent with a model in which the depolarization is due to the dissociation from and rotation of lipoic acids between binding sites on the multienzyme complex. The sum of the rate constants characterizing the association and dissociation with the binding sites is approximately 3 x 10(6) s-1. In addition, approximately 5% of the anisotropy of the N-(1-pyrenyl)maleimide-labeled complex decays with a rotational correlation time of 25 ns; this can be attributed to local motion of the probe. At high extents of N-(1-pyrenyl)maleimide labeling (90-95% inhibition of enzyme activity), the anisotropy decay can be described by a constant term plus a rotational correlation time of about 1 microseconds. The increase in the correlation time probably reflects interactions between pyrene moieties. The N-(1-pyrenyl)maleimide-labeled dihydrolipoyl transsuccinylase core of the multienzyme complex has been isolated, and the anisotropy is constant over the observed time range of 300 ns. This suggests that the native structure is necessary for observation of lipoic acid movement within the complex. Fluorescent-labeled limited trypsin digestion fragments of the alpha-ketoglutarate dehydrogenase complex also have been isolated, and anisotropy measurements reveal substantial mobility of the label within the fragments. The time-resolved anisotropy of FAD in the native complex and in the isolated dihydrolipoyl dehydrogenase indicates some rapid local mobility of the FAD (rotational correlation time of 12 ns) that is viscosity independent, as well as a component of the anisotropy that is constant over the 35-ns time scale of the experiments.
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PMID:Fluorescence polarization study of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli. 675 46

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 magnetic circular dichroic (MCD) spectra of oxidized and reduced flavins are recorded in various solvents. They are shown to be sensitive to flavin environment. The MCD spectra of oxidized and reduced lipoamide dehydrogenase are reported. In the oxidized enzyme the sign of the B term associated with the 27 000-cm-1 band is reversed from free flavins. This is attributed to interaction of the disulfide with the short-axis dipole of FAD. The sign reversal is also present in a closely related disulfide enzyme, glutathione reductase, but absent in glucose oxidase. In the half-reduced enzyme, the appearance of an A term at 18 180 cm-1 is attributable to a charge-transfer complex with a thiolate anion as donor. Insensitivity of the term's energy and intensity to the redox state of flavin suggests that a protein residue may accept or stabilize the thiolate charge transfer.
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PMID:Magnetic circular dichroism studies on the active-site flavin of lipoamide dehydrogenase. 689 75

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

Extensive amino acid sequence homology has been found between nine tryptic peptides of pig heart lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3] and the sequence of human erythrocyte glutathione reductase [NAD(P)H:glutathione oxidoreductase, EC 1.6.4.2]. The average homology is 40%. Six lipoamide dehydrogenase peptides are homologous with segments of the two parts of the FAD domain of glutathione reductase, one with the NADPH domain, and two with the interface domain. Thus, the homology extends throughout the molecule.
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PMID:Amino acid sequence homology between pig heart lipoamide dehydrogenase and human erythrocyte glutathione reductase. 695 34

Two-electron reduced glutathione reductase from yeast reacted with iodoacetamide is alkylated almost exclusively in the nascent thiol nearer the amino terminus of the protein. The charge-transfer absorbance, maximal at 530 nm, characteristic of the two-electron reduced enzyme is not lost as the alkylation proceeds, and the product has a spectrum virtually identical with that of the two-electron reduced enzyme. This observation demonstrates that the thiol alkylated is not the charge-transfer-donor thiolate which interacts with the FAD. The spectrum of the monoalkylated derivative is stable in the presence of oxidized glutathione, indicating that the charge-transfer-donor thiol is not involved in interchange with the substrate in the native enzyme. Thus, the nascent thiols produced upon two-electron reduction of glutathione reductase have distinct functions, interchange with the substrate and interaction with the FAD. Treatment of the monoalkylated derivative with the apolar phenylmercuric acetate eliminates the charge-transfer interaction. The spectrum of the resulting species is similar to that of the oxidized enzyme but less resolved and blue shifted by 10 nm. The dependence on pH of the absorbance associated with the thiolate to FAD charge-transfer interaction in native two-electron reduced glutathione reductase is biphasic, with pK values at approximately 4.8 and 7.4. By analogy with glyceraldehyde-3-phosphate dehydrogenase and papain, these data indicate that the thiolate is stabilized by an adjacent basic residue. The pK 7.4 is associated with the titration of the base to give the ion pair, and the pK of 4.8 is associated with the titration of the thiolate. Unlike lipoamide dehydrogenase, glutathione reductase is sufficiently stable to allow titration with dithionite at pH 3.7. The spectrum at this pH is essentially the same as that of the monoalkylated derivative treated with phenylmercuric acetate. The changes with pH are completely reversible.
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PMID:Glutathione reductase from yeast. Differential reactivity of the nascent thiols in two-electron reduced enzyme and properties of a monoalkylated derivative. 701 96

Glutathione reductase (Mr 2 x 52 500), a flavoenzyme of known three-dimensional structure, catalyses the reduction of glutathione disulfide by NADPH. This paper describes the primary structure of the FAD-binding domain which ranges from AcAla-1 to Gly-157. The three CNBr-produced fragments (69, 10 and 80 residues) of the domain were fractionated further by enzymatic and chemical methods; isolated peptides were sequenced mainly by automatic solid-phase Edman degradation. The tryptic peptides were overlapped by chymotryptic peptides. A fragment which results from cleavage at the acid-labile bond between Asp-135 and Pro-136 supplied peptides for overlapping the CNBr-produced fragments. In addition, many peptides were ordered and overlapped by computerized comparison with a complete sequence guessed from the electron density map. With one exception the computer method and the chemical alignment gave the same results. The sequence data are discussed in the light of the secondary and tertiary structure (Schulz et al. (1978) Nature (Lond.) 273, 120--124]. The 17 N-terminal residues are not visible in the electron density map. Consequently our numbering scheme differs from that of Schulz et al. by approximately 20 residues. Acetylation of the N terminus and an unusual composition of the following residues may serve to protect the loose N-terminal section of the protein against proteolysis in situ. The four cysteinyl residues of the FAD domain are of special interest. Cys-2 at the tip of the N-terminal extension is likely to be involved in the aggregation behaviour of glutathione reductase. Cys-58 and Cys-63 (formerly Cys-41 and Cys-46) represent the enzyme's redox-active dithiol. Cys-90 with its location at the twofold axis forms a disulfide bridge with Cys-90 of the other peptide chain of the enzyme. This might be related to the fact that both peptide chains contribute to each of the two active centers. In view of the interchain disulfide bridge glutathione reductase should be regarded as a monomeric protein. The sequence of the FAD-binding domain was compared with the sequence of the NADPH-binding domain of glutathione reductase using a computer program. As discussed, the scarcity of sequence similarities does not argue against the assumption that the two nucleotide-binding domains of glutathione reductase originated by gene duplication. The pyrophosphate moiety of FAD binds to a part of the polypeptide chain which in geometric structure, in topology and in sequence resembles the phosphate loops of other nucleotide-binding proteins and of flavodoxin. Using the phosphate loop as a reference, the N-terminal sequence of five flavoproteins can be aligned. The results of Williams et al. on the sequence of lipoamide dehydrogenase (EC 1.6.4.3) and our data on glutathione reductase (EC 1.6.4.2) show clearly that these two mechanistically similar enzymes possess homologous structures.
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PMID:Glutathione reductase from human erythrocytes: amino-acid sequence of the structurally known FAD-binding domain. 703 15

1. Sequence analysis of the NADPH domain (residues 158--293) and of the interface domain (365--478) was based on 12 CNBr fragments, which were isolated using ion-exchange chromatography and paper methods. Fragments with more than 15 residues were digested further with trypsin and chymotrypsin. The isolated peptides were sequenced by automated solid-phase Edman degradation. All sequenced peptides were ordered and overlapped by computerized comparisons with a complete sequence guessed from the electron density map of the protein. In the case of short CNBr fragments, this alignment was confirmed by the sequence analysis of protein fragments resulting from incomplete CNBr cleavage. 2. In the NADPH domain, residue 197, which is involved in an induced-fit mechanism, was identified as a tyrosine. The structure of the NADPH domain is probably homologous with the NAD domain of lipoamide dehydrogenase and with the FAD domain of several proteins, but not with NADPH domains of known chain-fold in other proteins. 3. The paper completes the sequence analysis of glutathione reductase so that the enzyme is now known in atomic detail. The numbering scheme of the chemically determined sequence will be used henceforth in crystallographic studies also. As inferred from the sequence data each of the two identical chains contains 478 amino acid residues, the composition being Cys10, Asp21, Asn17, Thr31, Ser31, Glu29, Gln11, Pro24, Gly43, Ala42, Val44, Met15, Ile29, Leu34, Tyr13, Phe14, Lys34, His16. Arg17, and Trp3. From these data an Mr of 2 x 51 600 was calculated for the FAD-free apoenzyme and an Mr of 2 x 42 400 for the holoenzyme.
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PMID:Glutathione reductase from human erythrocytes. The sequences of the NADPH domain and of the interface domain. 706 May 51


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