UMLS:C0027960 - FACTA Search

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Query: UMLS:C0027960 (mole)
21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Salicylate hydroxylase from Pseudomonas putida S-1 was irreversibly inactivated by trinitrobenzenesulfonic acid (TNBS). The reaction was linearly dependent on TNBS concentration and the second-order rate constant was 120 M-1.min-1 for the holoprotein at pH 8.5. Modification of one mole of lysine residue per mole of enzyme caused a large loss of the activity, and the enzyme was no longer able to show NADH-dehydrogenase activity after uncoupling. The presence of NADH, NAD+, ATP, or AMP afforded protection against the inactivation. The enzyme modified at a single lysine residue was isolated by hydrophobic chromatography as an apoprotein form and characterized. It could bind FAD with the same Kd value for that of native apoprotein. The apparent Michaelis constant of the enzyme was increased 13-fold for NADH, but not for salicylate. Vmax for NADH oxidation was decreased to one-fifth of that of the native enzyme. A peptide containing one trinitrophenyl-lysine residue was isolated from the chymotryptic digest of the modified enzyme and its amino acid sequence was determined to be TADVAIAADGIKSSM, which is homologous to the sequence from R-154 to I-168 of salicylate hydroxylase from P. putida PpG7. The lysine in the peptide may represent a basic residue interacting with an anionic group of NADH in the binding site of the enzyme.
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PMID:Identification of a lysine residue in the NADH-binding site of salicylate hydroxylase from Pseudomonas putida S-1. 762 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

Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channelling maleylacetate and some chlorinated derivatives into the 3-oxoadipate pathway. Several substituted maleylacetates were prepared in situ by alkaline or enzymatic hydrolysis of dienelactones as the precursor. The conversion of these methyl-, chloro-, fluoro-, and bromo-substituted maleylacetates by malelacetate reductase from 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. Two moles of NADH per mole of substrate was consumed for the conversion of maleylacetates which contain a halogen substituent in the 2 position. In contrast, only 1 mol of NADH was necessary to convert 1 mol of substrates without a halogen substituent in the 2 position. The conversion of 2-fluoro-, 2-chloro-, 2,3-dichloro-, 2,5-dichloro-, 2,3,5-trichloro-, 2-bromo-, 2,3-dibromo-, 2,5-dibromo-, 2-bromo-5-chloro-, 2-chloro-3-methyl-, and 2-chloro-5-methylmaleylacetate was accompanied by the elimination of halide from the 2 position and the temporary occurrence of the corresponding dehalogenated maleylacetate as an intermediate consuming the second mole equivalent of NADH. The properties of the halogen substituents influenced the affinity to the enzyme in the following manner. Km values increased with increasing van der Waals radii and with decreasing electronegativity of the halogen substituents (i.e., low steric hindrance and high electronegativity positively influenced the binding). The Km values obtained with 2-methyl-,3-methyl-, and 5-methylmaleylacetate showed that a methyl substituent negatively affected the affinity in the following order: 2 position >/ = 3 position >> 5 position. A reaction mechanism explaining the exclusive elimination of halogen substituents from the 2 position is proposed.
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PMID:Maleylacetate reductase of Pseudomonas sp. strain B13: specificity of substrate conversion and halide elimination. 781 20

Seven chimeric biomimetic dye-ligands (BM) are purpose-designed and synthesized by specific structural modification of the parent anthraquinone dichlorotriazine dye Vilmafix blue A-R (VBAR). Each BM dye is composed of two enzyme-recognition moieties. The terminal biomimetic moiety bears a variable carboxylated structure linked to the triazine ring, thus mimicking the substrate of formate dehydrogenase (FDH). The anthraquinone moiety remains the same as that of the parent dye and recognizes the nucleotide-binding area of the target enzyme. Dyes are purified by liquid column chromatography (typically 99%), analyzed by liquid-paper chromatography, thin-layer chromatography, and high-performance liquid chromatography, and their lambda max and epsilon values are determined. The ability of dyes to act as affinity ligands versus Candida boidinii FDH is evaluated by kinetic studies and determining KD values from both difference spectra and enzyme inactivation studies. The parent dichlorotriazine dye VBAR binds specifically and irreversibly to FDH (k3 0.19 min-1; KD 19.3 microM). The inactivation of the NAD(+)-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH, and ADP. Quantitatively inhibited FDH contained approx 1 mol of dye per mole of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM each of NAD+, NADH, and ADP or by extensive dialysis or gel filtration chromatography. The monochlorotriazine BM dyes do not inactivate FDH but inhibit competitively the inactivation by VBAR. When compared to VBAR and Cibacron blue 3GA (CB3GA), all BM dye-ligands exhibited lower KD values. FDH generally preferred binding to BM ligands which bore an aromatic terminal biomimetic moiety substituted with a monocarboxyl group rather than an alpha-ketoacid. Dye binding to FDH is accompanied by a characteristic spectral change in the range 550-800 nm. This phenomenon is perturbed after titration by increasing amounts of NAD+. Electrostatic interactions appeared to play a dominant role in the dye.FDH complex. The BM dye-ligand bearing a m-aminobenzoate at its terminal biomimetic moiety (BM1) exhibited the highest affinity (KD 1.6 microM, 8.0-fold decrease over CB3GA). BM1 differentiated between the binding sites of FDH, displaying uncompetitive inhibition with respect to NAD+ (Ki 15.6 microM) and competitive with respect to formate (Ki 18.1 microM).
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PMID:The interaction of Candida boidinii formate dehydrogenase with a new family of chimeric biomimetic dye-ligands. 784 Jun 13

The tetrameric mung bean glyceraldehyde-3-phosphate dehydrogenase is found to bind approximately four moles substrate, glyceraldehyde-3-phosphate, per mole enzyme with Kdiss equal to or less than 9.6 microM at pH 7.3, showing a slight positive cooperativity. Addition of excess substrate to a solution of the enzyme and excess NAD+ leads to a "burst" of NADH formation followed by a slow linear increase (monitored spectrophotometrically). Amount of NADH formed in the burst phase is pH-dependent and is equal to 3.6 moles per mole enzyme at pH 8.6 and above. Presuming four equivalent and independent sites per enzyme molecule (i.e. D2-symmetry), consistent values were obtained for the equilibrium constant of the oxidation-reduction step at different pH and most substrate concentrations. At lower pH (7.3) and high [NAD+]/[substrate] ratios, favouring the C2- symmetry conformation of the enzyme, the magnitude of the burst phase was negligibly small; practically no oxidation reduction reaction took place. Combining these with earlier results on the group transfer step, it is suggested that the oxidation-reduction and group transfer steps of the reaction catalysed by this enzyme require the D2 and C2 symmetry conformations of the enzyme, respectively.
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PMID:Functional significance of protein conformational isomerisation in the glyceraldehyde-3-phosphate dehydrogenase-catalysed reaction. 811 17

The electron carriers of the mitochondrial NADH:ubiquinone oxidoreductase (complex I) are contained predominately in two extramembranous subcomplexes, a flavoprotein (FP) and an iron-sulfur protein (IP). FP contains three subunits with molecular masses of 51, 24, and 9 kDa. The 51-kDa subunit carries the NADH binding site and contains FMN and a tetranuclear iron-sulfur cluster. The 24-kDa subunit contains a binuclear iron-sulfur cluster. IP contains seven subunits with molecular masses of 75, 49, 30, 18, 15, 13, and 11 kDa. It contains a tetranuclear and very likely a binuclear iron-sulfur cluster in the 75-kDa subunit. FP and IP make contact through the 51- and the 75-kDa subunits. The remainder of complex I (hydrophobic protein (HP), 31 subunits) is largely membrane-intercalated and contains two iron-sulfur clusters apparently in a 23-kDa subunit and possibly another in a 20-kDa subunit. In this study, the stoichiometries of the FP and IP subunits in complex I were determined by radioimmunoassay. Per mole of complex I, there are 2 mol of the 15-kDa subunit and 1 mol each of the FP and the four largest IP subunits. The stoichiometries of the 13- and the 11-kDa subunits could not be determined separately, because they comigrate upon gel electrophoresis. In addition, the effect of substrates (NADH, NADPH, NAD, and NADH plus potassium ferricyanide to rapidly oxidize NADH via FP) on the cross-linking patterns of FP and IP subunits was investigated, using three different cross-linking reagents of different molecular lengths.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catalytic sector of complex I (NADH:ubiquinone oxidoreductase): subunit stoichiometry and substrate-induced conformation changes. 816 12

The contractile protein actin contains one mole of firmly bound nucleotide and a number of divalent cations bound with different affinities. During recent years evidence for a second nucleotide interacting site on actin has been reported. Therefore, a specific search for the presence of a second nucleotide-interacting site on actin was undertaken. For this purpose G- and F-actin or actin in complex with deoxyribonuclease I (DNase I) was passed over ADP-agarose which was found to retain all three forms of actin. Nucleotide bound to the high affinity site of actin did not exchange during passage and retention to agarose-immobilized ADP, thus indicating the presence of a second nucleotide interacting site. This site was found to be equally accessible in G- and F-actin and in the actin-DNase I complex, whereas DNase I alone passed unretained through this column. A number of nucleotides and phosphorylated compounds were tested for their ability to compete with immobilized ADP for actin interaction. It was found that all forms of actin are liberated only by high concentrations (5mM) of ADP, ATP and NADH, by 1mM CTP and ITP, and by high salt concentrations (150mM NaCl). Since it was found that EDTA- and heat-treated actin were also retained on ADP-agarose, we conclude that this second nucleotide interacting site is of limited specificity, low affinity, and not dependent on the native configuration of actin. It exhibits characteristics of an unspecific, polyanionic site, but may represent the low affinity phosphate binding site.
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PMID:Evidence that the presumptive second nucleotide interacting site on actin is of low specificity and affinity. 848 39

A low-cost assay method that is able to measure H2O2 concentrations as low as the nano-molar range is described. The assay solution contains NADH, horseradish peroxidase, and superoxide dismutase at PH 7.5. After the addition of the sample, the decrease in NADH concentration measured by spectrophotometry is proportional to the H2O2 concentration. Because of superoxide dismutation, a high amplification factor defined as moles NADH oxidised per mole H2O2 added is obtained, which allows the sensitivity limit of the method to be greatly improved. We have established the conditions under which the amplification factor can be stabilised at a high level: the best compromise is to increase both the horseradish peroxidase and superoxide dismutase concentrations. Finally, we have also shown that coupled to specific oxidases, our assay method is suitable for measuring very low concentrations of biochemicals that can be oxidized by oxygen with H2O2 production.
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PMID:Experimental procedure for a hydrogen peroxide assay based on the peroxidase-oxidase reaction. 870 81

Interactions of NAD-dependent dehydrogenases (glyceraldehyde-3-phosphate dehydrogenase, GAPDH, and lactate dehydrogenase, LDH) with band 3 erythrocyte membrane protein and tubulin were characterized. At low ionic strength and un-saturating substrate concentrations, LDH tightly binds to tubulin and is thus inactivated. The Kd of the LDH-tubulin complex was calculated in inhibition and direct binding experiments (15.0 and 13.6 nM, respectively); the stoichiometry of the complex was 1.66 moles of tubulin dimer bound per mole of LDH tetramer. In the presence of 0.15 M NaCl, LDH does not bind to tubulin and tubulin-dependent inhibition of LDH activity is not detected. At low ionic strength, erythrocyte membranes affect both dehydrogenases similarly. GAPDH activity is completely inhibited by excess of erythrocyte membranes (or by excess of cytoplasmic fragment of band 3 protein). Under similar conditions, LDH activity was inhibited by 70% by erythrocyte membranes. In these experiments, 14.8.10(6) GAPDH tetramers or 25.6.10(6) LDH tetramers bound to one erythrocyte ghost (Kd is 0.13 and 0.6 microM, respectively). Increase in ionic strength (0.15 m NaCl) completely abolished the membrane-dependent inhibition of dehydrogenases; however, membranes still bound GAPDH and LDH. Under these conditions, the Kd for GAPDH was increased (up to 4.43 microM), whereas the number of membrane-bound enzyme molecules has not been significantly affected (0.75 nmoles of tetramer per 100 micrograms membrane protein). The Kd for LDH was not changed (0.76 microM), whereas the number of membrane-bound enzyme molecules was decreased (down to 0.48 nmoles of tetramer per 100 micrograms membrane protein). It is suggested that at low ionic strength, the "acidic tails" of band 3 protein and tubulin can interact with positively charged NAD-binding domains of both dehydrogenases thus inhibiting their activity. Increase in ionic strength reduces these interactions, decreasing the binding and inhibition of enzyme activities. At "physiological" ionic strength, catalytically active GAPDH and LDH can possibly bind to various sites of the erythrocyte membrane. This can be important in regulation of the transfer of the common cofactor (NAD/NADH) between their active sites.
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PMID:[Effect of erythrocyte membranes and tubulin on the activity of NAD-dependent dehydrogenases]. 896 25

Cells of Saccharomyces cerevisiae were permeabilized by ether for the isolation of coenzyme NADH. A 4-fold increase in the ether fraction to aqueous fraction resulted in the recovery of 80% of total NADH present in the cell. NADH was separated and purified by affinity ultrafiltration using yeast alcohol dehydrogenase as an affinity ligand. The binding characteristics of the enzyme and coenzyme were established at different pH and ionic strengths using gel filtration. The number of moles of NADH bound per mole of alcohol dehydrogenase (r) was found to be 5.7 at pH 8 and ionic strength (I) 0.1 M. The binary complex of NADH and alcohol dehydrogenase was cleaved by lowering the pH to 6.0. The crude cell permeate on purification by ultrafiltration with 2-fold dilution, gave NADH with an absorbance ratio (A260/A340) of 2.3 and overall yield of 68%. Alcohol dehydrogenase was recovered as retentate with 93% recovery and 15% loss in activity.
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PMID:Isolation of NADH from Saccharomyces cerevisiae by ether permeabilization and its purification by affinity ultrafiltration. 905 54

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