Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
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Indoleamine 2,3-dioxygenase was purified from rabbit small intestine to apparent homogeneity as judged by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The native enzyme was a monomeric protein of a molecular weight of 41,000 +/- 1,000 with an s020,w value of 3.45 S. It had a relative abundance of hydrophobic amino acids such as valine, leucine, and isoleucine, and contained approximately 5% carbohydrate by weight. The estimated content of sugar residues per mol of enzyme was: galactose, 1.2; mannose, 2.6; N-acetylglucosamine, 5.2; and sialic acid, 0.8. One mole of enzyme had 0.8 mol of protoheme IX as a prosthetic group. However, copper was not detected in a significant amount and the ratio of copper to heme was less than 0.03. EPR spectra of the nitric oxide complex of the ferrous enzyme indicated that a nitrogen atom, possibly in an imidazole group, might be coordinated as the fifth ligand of the heme coenzyme. The anisotropic g values were gx = 2.08, gy = 1.98, and gz = 2.01. A single enzyme protein catalyzed the oxygenative ring cleavage of D- and L-tryptophan, D- and L-5-hydroxytryptophan, tryptamine, and serotonin. In addition, the purified enzyme had a peroxidase activity with guaiacol and potassium iodide as hydrogen donors, but not a catalase activity.
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PMID:Indoleamine 2,3-dioxygenase. Purification and some properties. 2 87

1) It was demonstrated by colorimetric as well as EPR measurements that the native (aerobic, resting state) Rhus vernicifera laccase contains both Cu2+ and Cu+ (total Cu content was 4.0 gram atoms/mole). The ratio of Cu2+ to total Cu in laccase varied (42-90%) in samples of latex collected from various districts. The absorption maximum at 615 nm was proportional to the content of total Cu in the enzyme sample. Laccase activity was found to almost parallel the content of the Cu2+ form. The oxidized minus reduced difference absorbance of the enzyme at 330 nm shoulder was proportional to the amount of Cu2+. 2) Steady state level of oxidation of laccase copper during the laccase copper catalytic action, the rates of reduction by substrates and the oxidation by O2 were determined by following absorbance changes at 615 and 330 nm by the stopped flow method. 3) All the results from titrimetric and kinetic experiments were consistent with the laccase model previously proposed by Makino and Ogura in which a laccase molecule contains 1 Cu(615) and 3 Cu(330). Our expanded model states that a laccase sample originally contains active as well as inactive enzymes. In the active enzyme, Cu ions are reactive to O2 but in the inactive enzyme, Cu can be oxidized only by oxidizing agents such as H2O2 or ferricyanide, or by a slow intermolecular electron transfer from Cu(615) to the active enzyme. In both species of enzyme rapid reduction of Cu2+ ions by substrate takes place. In comparative studies of the reactivities of Cu ions in various copper proteins, we would like to suggest that oxidatic activity of a copper protein is due to the Cu+ form of the enzyme ions with O2.
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PMID:Oxidation and reduction of copper ions in catalytic reactions of Rhus laccase. 13 27

1. Titration of Neurospora tyrosinase with 2-mercaptoethanol shows that the increase of absorbance at 700 nm is directly correlated to the loss of enzymatic activity. Approximately 2 mol of 2-mercaptoethanol per mole of protein are needed for full development of the green, enzymatically inactive complex. The increase of absorbance at 700 nm is also proportional to the intensity of the EPR signal and the amount of non-covalently bound 2-[35S] mercaptoethanol to the enzyme. The maximal EPR intensity reaches 70% of the protein concentration and at most 0.7--0.8 mol of 2-[35S] mercaptoethanol is bound per mol of enzyme. 2. Stopped-flow measurements show that in the reaction between 2-mercaptoethanol and Neurospora tyrosinase a raction intermediate with a strong absorption band at 360 nm is formed in an apparent second-order reaction. This intermediate displays no EPR-detectable signals. The intermediate decays in a similar complex fashion as the absorption band at 700 nm is formed. 3. The reaction of Neurospora tyrosinase with a variety of sulfhydryl compounds was also investigated. In most cases green coloured, enzymatically inactive complexes are formed displaying slightly different EPR signals. However, with cysteine and cysteamine violet coloured, enzymatically inactive complexes are formed which show rather different EPR signals. The integrated EPR intensities amount to 40--70% of the protein concentration. Based on simulations of 9 and 35 GHz spectra all observed EPR spectra can be represented as true S = 1/2 systems. The cysteamine complex can be interpreted as arising from a mixed valence Cu2+ . Cu+ complex. The 2-mercaptoethanol spectra can, however, arise from sulphur radicals. 4. Treatment of Agaricus bispora tyrosinase and Cancer pagures hemocyanin with 2-mercaptoethanol results in green-coloured, EPR detectable complexes similar to the one found with Neurospora tyrosinase. No such complexes are formed when hemocyanins from Helix pomatia and Panulirus interruptus were treated with this reagent.
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PMID:The reaction of mercaptans with tyrosinases and hemocyanins. 20 26

A thenoyl trifluoroacetone-sensitive and antimycin-insensitive ubisemiquinone radical (Qs) is readily detected in purified succinate-cytochrome c reductase. When this reductase is resolved into succinate-Q and ubiquinol-cytochrome c reductases, Qs was not detected in either reductase. The difficulty in detecting such a radical in purified succinate-Q reductase has puzzled investigators for years. A deficiency of Q in the isolated complex is the reason for the failure to detect Qs. Upon addition of exogenous Q, a thenoyl trifluoroacetone-sensitive Q-radical is readily detectable in isolated succinate-Q reductase under a controlled redox potential. Maximum radical concentration is observed when 5 mol of exogenous Q, per mole of flavin, is added. The radical gives an EPR signal with a g-value of 2.005 and a line-width of 12 G. The Em of Qs is 84 mV at pH 7.4, with half-potentials of E1 = 40 mV and E2 = 128 mV. The Qs-radical does not show power saturation, even at 200 mW.
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PMID:Characterization of ubisemiquinone radicals in succinate-ubiquinone reductase. 130 86

A soluble [NiFe] hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile. A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21. This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa. This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction. The H2/HD ratio is lower than one in the D2-H+ exchange reaction. The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2-. The EPR spectrum of the native hydrogenase shows the presence of a [3Fe-4S] oxidized cluster and of a Ni(III) species.
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PMID:Partial purification and characterization of the first hydrogenase isolated from a thermophilic sulfate-reducing bacterium. 131 68

Redox titrations of carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum were performed using the reductant CO and the oxidant thionin. Titrations were followed at 420 nm, a wavelength sensitive to redox changes of the iron-sulfur clusters in the enzyme. When CODH was oxidized by just enough thionin to maximize A420, two molecules of CO per mole of CODH dimer (4 equiv/mol) reduced the enzyme fully. Likewise, 4 equiv/mol of thionin oxidized the fully-reduced enzyme to the point where A420 maximized. The four n = 1 redox sites which titrated in this region were designated group I sites. They include at least two iron-sulfur clusters, [Fe/S]A and [Fe/S]B, and two other sites, A' and B'. The [Fe4S4]2+/1+ cluster in CODH is included in this group. [Fe/S]B and B' have reduction potentials (at pH 8) below -480 mV vs NHE; [Fe/S]A and A' have reduction potentials above that value. The reduction potential of either [Fe/S]B or B' is near to the CO/CO2 couple at pH 8 (-622 mV). When CODH was oxidized by more than enough thionin to maximize A420, some of the excess thionin oxidized the so-called group II redox sites. These sites have reduction potentials more positive than group I and do not exhibit changes at 420 nm when titrated. Titration of group II sites required 1-2 equiv/mol. EPR of reduced group II sites exhibited the gav = 1.82 signal. When these sites were oxidized, the only signal present had g values at 2.075, 2.036, and 1.983.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Redox titrations of carbon monoxide dehydrogenase from Clostridium thermoaceticum. 132 Sep 27

It has been shown previously that Escherichia coli contains three fumarase genes designated fumA, fumB, and fumC. The gene products fumarases A, B, and C have been divided into two classes. Class I contains fumarases A and B, which have amino acid sequences that are 90% identical to each other, but have almost no similarity to the sequence of porcine fumarase. Class II contains fumarase C and porcine fumarase, which have amino acid sequences 60% identical to each other [Woods, S.A., Schwartzbach, S.D., & Guest, J.R. (1988) Biochim. Biophys. Acta 954, 14-26]. In this work it is shown that purified fumarase A contains a [4Fe-4S] cluster. This conclusion is based on the following observations. Fumarase A contains 4 Fe and 4 S2- per mole of protein monomer. (The mobility of fumarase A in native polyacrylamide gel electrophoresis and the elution volume on a gel permeation column indicate that it is a homodimer.) Its visible and circular dichroism spectra are characteristic of proteins containing an Fe-S cluster. Fumarase A can be reduced to an EPR active-state exhibiting a spectrum consisting of a rhombic spectrum at high fields (g-values = 2.03, 1.94, and 1.88) and a broad peak at g = 5.4. Upon addition of substrate, the high field signal shifts upfield (g-values = 2.035, 1.92, and 1.815) and increases in total spins by 8-fold, while the g = 5.4 signal disappears.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fumarase a from Escherichia coli: purification and characterization as an iron-sulfur cluster containing enzyme. 132 45

Tri-n-butyltin(TBT) compounds having the chemical formula (C4H9)3SnX are broad-spectrum biocidal agents whose toxic effect is primarily at the membrane level. Red blood cells (RBC) exposed to micromolar concentrations of tributyltin compounds (TBTX) undergo morphological changes and hemolysis. Determination of the mechanism of action whereby TBT elicits membrane damage continues to be a challenging endeavor. Because xenobiotic TBT+ and endogenous O2.- have been found to penetrate and alter RBC membrane function, it is hypothesized that they may combine chemically within the RBC hydrophobic lipid bilayer during TBT insult to initiate lipid peroxidative processes. The present study has been designed (1) to determine if TBT+ and O2.- combine chemically in aprotic media and, if so, (2) to characterize any free-radical complex(es) generated. The reactions of the membrane-active TBTX compounds (X = OCH3, Cl, Br, or I) with O2.- have been investigated in the aprotic solvent system cis-dicyclohexano-18-crown-6 ether/DMSO using EPR techniques. When incremental amounts of each TBT halide were added to O2.- solutions at room temperature, the EPR signal characteristic of O2.- diminished in intensity and disappeared when a 1:1 O2.-/TBT+ mole ratio was attained. Although the same phenomenon was observed for all TBT halides used, only the KO2/TBTI reaction produced detectable amounts of a new oxygen-centered free-radical complex. The EPR spectral parameters calculated from the product anisotropic frozen glass spectra were gx = 2.054, a(x) = 31.7 G, gy = 2.021, gz = 2.002, gav = 2.026.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:In vitro activation of lipophilic tributyltins by superoxide produces tributylstannyl superoxo radicals, proposed initiators of lipid peroxidation: an EPR model study. 133 86

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.
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PMID:An active-site lysine in avian liver phosphoenolpyruvate carboxykinase. 190 75

A ferredoxin has been purified from Streptomyces griseus grown in soybean flour-containing medium. The homogeneous protein has a molecular weight near 14,000 as determined by both PAGE and size exclusion chromatography. The iron and labile sulfide content is 6-7 atoms/mole protein. EPR spectroscopy of native S. griseus ferredoxin shows an isotropic signal at g = 2.01 which is typical of [3Fe-4S]1+ clusters and which quantitates to 0.9 spin/mole. Reduction of the ferredoxin by excess dithionite at pH 8.0 produces an EPR silent state with a small amount of a g = 1.95 type signal. Photoreduction in the presence of deazaflavin generates a signal typical of [4Fe-4S]1+ clusters at much higher yields (0.4-0.5 spin/mole) with major features at g-values of 2.06, 1.94, 1.90 and 1.88. This latter EPR signal is most similar to that seen for reduced 7Fe ferredoxins, which contain both a [3Fe-4S] and [4Fe-4S] cluster. In vitro reconstitution experiments demonstrate the ability of the S. griseus ferredoxin to couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450soy for NADPH-dependent substrate oxidation. This represents a possible physiological function for the S. griseus ferredoxin, which if true, would be the first functional role demonstrated for a 7Fe ferredoxin.
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PMID:Purification and characterization of a 7Fe ferredoxin from Streptomyces griseus. 215 56


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