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)

Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi, the causative agent of Chagas' disease, was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. LADH lipoamide reductase and diaphorase activities decreased as a function of incubation time and composition of the MPO/H2O2/halide system, a transient increase preceding the loss of diaphorase activity. Iodide, bromide, thiocyanide and chloride were effective components of MPO/H2O2 or MPO/NADH systems. Catalase prevented LADH inactivation by the MPO/NADH/halide systems in agreement with H2O2 production by NADH-supplemented LADH. Thiol compounds (L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine) and Captopril prevented LADH inactivation by the MPO/H2O2/NaCl system and by NaOCl, thus supporting HOCl as agent of the MPO/H2O2/NaCl system. MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 inactivated LADH, the reaction being prevented by MPO inhibitors and thiol compounds. T. cruzi LADH was affected by MPO-dependent systems like myocardial LADH, allowance being made for the variation of the diaphorase activity and the greater sensitivity of the T. cruzi enzyme to MPO/H2O2/halide systems.
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PMID:Inactivation of Trypanosoma cruzi dihydrolipoamide dehydrogenase by leukocyte myeloperoxidase systems: role of hypochloride and nitrite related radicals. 1100 5

The thioredoxin redox system is composed of the NADPH-dependent homodimeric flavoprotein thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin. It is responsible for the reduction of disulfide bridges in proteins such as ribonucleotide reductase and several transcription factors. Furthermore, thioredoxin is involved in the detoxification of hydrogen peroxide and protects the cell against oxidative damage. There exist two classes of TrxRs: the high M(r) and the low M(r) proteins. The well characterized Escherichia coli TrxR represents a member of the low M(r) class of proteins, whereas the mammalian, Caenorhabditis elegans, and Plasmodium falciparum proteins belong to the family of high M(r) proteins. The primary structure of these proteins is very similar to that of glutathione reductase and lipoamide dehydrogenase. However, the high M(r) TrxRs possess, in addition to their redox active N-terminal pair of cysteines, a pair of cysteine residues or a selenenylsulfide motif at their C terminus. These residues have been shown to be crucial for the reduction of thioredoxin. In this study we address the question whether the active site residues of P. falciparum TrxR are provided by one or both subunits. Differentially tagged wild-type and PfTrxR mutants were co-expressed in E. coli and the recombinant protein species were purified by affinity chromatography specific for the respective tags of the recombinant proteins. Co-expression of PfTrxR wild-type and mutant proteins resulted in the formation of three different protein species: homodimeric PfTrxR wild-type proteins, homodimeric mutant proteins, and heterodimers composed of one PfTrxR wild-type subunit and one PfTrxR mutant subunit. Co-expression of the double mutant PfTrxRC88AC535A with PfTrxR wild-type generated an inactive heterodimer, which indicates that PfTrxR possesses intersubunit active sites. In addition, the data presented possibly imply a coopertive interaction between both active sites of PfTrxR.
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PMID:Intersubunit interactions in Plasmodium falciparum thioredoxin reductase. 1102 50

Thiol-containing compounds, such as glutathione and cysteine, react with selenite under specific conditions to form selenotrisulfides. Previous studies have focused on isolation and characterization of intermolecular selenotrisulfides. This study describes the preparation and characterization of intramolecular selenotrisulfide derivatives of lipoic acid and lipoamide. These derivatives, after separation from other reaction products by reverse-phase HPLC, exhibit an absorbance maximum at 288 nm with an extinction coefficient of 1,500 M(-1) small middle dotcm(-1). The selenotrisulfide derivative of lipoic acid was significantly stable at or below pH 8.0 in contrast to several other previously studied selenotrisulfides. Mass spectral analysis of the lipoic acid and lipoamide derivatives confirmed both the expected molecular weights and also the presence of a single atom of selenium as revealed by its isotopic distribution. The selenotrisulfide derivative of lipoic acid was found to serve as an effective substrate for recombinant human thioredoxin reductase as well as native rat thioredoxin reductase in the presence of NADPH. Likewise, the lipoamide derivative was efficiently reduced by NADH-dependent bovine lipoamide dehydrogenase. The significant in vitro stability of these intramolecular selenotrisulfide derivatives of lipoic acid can serve as an important asset in the study of such selenium adducts as model selenium donor compounds for selenophosphate biosynthesis and as rate enhancement effectors in various redox reactions.
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PMID:Synthesis and characterization of selenotrisulfide-derivatives of lipoic acid and lipoamide. 1105 Jan 72

1. Addition of Cr VI (dichromate) to isolated rat hepatocytes results in rapid glutathione oxidation, reactive oxygen species (ROS) formation, lipid peroxidation, decreased mitochondrial membrane potential and lysosomal membrane rupture before hepatocyte lysis occurred. 2. Cytotoxicity was prevented by "ROS" scavengers, antioxidants, and glutamine (ATP generator). Hepatocyte dichlorofluorescin oxidation (to determine ROS/Cr V formation) was inhibited by mannitol (a hydroxyl radical scavenger) or butylated hydroxyanisole and butylated hydroxytoluene (antioxidants). 3. The Cr VI reductive mechanism required for toxicity are not known. Cytotoxicity was also prevented by cytochrome P450 inhibitors, particularly CYP 2E1 inhibitors, but not inhibitors of DT diaphorase or glutathione reductase. This suggests that P450 reductase and/or reduced cytochrome P450 contributes to Cr VI reduction to Cr IV. 4. Glutathione depleted hepatocytes were resistant to Cr (VI) toxicity and much less dichlorofluorescin oxidation occurred. Reduction of dichromate by glutathione or cysteine in vitro was also accompanied by oxygen uptake and was inhibited by Mn II (a Cr IV reductant ). Cr VI induced cytotoxicity and ROS formation was also inhibited by Mn II which suggests that Cr IV and Cr IV.GSH mediate "ROS" formation in isolated hepatocytes. 5. In conclusion Cr VI cytotoxicity is associated with mitochondrial/lysosomal toxicity by the biological reactive intermediates Cr IV and "ROS".
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PMID:Biological reactive intermediates that mediate chromium (VI) toxicity. 1176 36

Peroxidase/H2O2/phenothiazine systems irreversibly inhibit Trypanosoma cruzi dihydrolipoamide dehydrogenase (LADH). Inactivation of the parasite enzyme depended on (a) phenothiazine structure; (b) peroxidase nature; (c) incubation time and (d) the presence of a cation radical scavenger. With the myeloperoxidase/H2O2/system, promazine, trimeprazine, thioridazine, promethiazine, prochlorperazine, chlorpromazine and perphenazine were the most effective derivatives out of twelve phenothiazines studied. An electronegative substituent at position 2 of the phenothiazine ring such as Cl, or trifluoromethyl, propionyl and nitrile groups decreased or nullified phenothiazine activity. Myeloperoxidase/H2O2/, horseradish peroxidase/H2O2/, and myoglobin/H2O2/systems activated phenothiazines producing the corresponding cation radicals, myeloperoxidase being the most selective one with respect to phenothiazine structure. The myoglobin/H2O2/system activated phenothiazines that were scarcely active or inactivate with the MPO/H2O2/system, such as the trifluoromethyl derivatives. Production of phenothiazine cation radicals was demonstrated by optical spectroscopy. Phenothiazine cation radical stability depended on their structure as illustrated by promazine and thioridazine. Thiol compounds (GSH, N-acetyl-cysteine and penicillamine), aromatic aminoacids (L-tyrosine, L-tryptophan, and the corresponding peptides) and ascorbate scavenged phenothiazine cation radicals, thus preventing LADH inactivation. Comparison of the summarized phenothiazine effects with those of phenothiazines on T. cruzi suggest the role of cation radicals in phenothiazines chemotherapeutic actions.
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PMID:Myeloperoxidase-generated phenothiazine cation radicals inactivate Trypanosoma cruzi dihydrolipoamide dehydrogenase. 1218 Feb 62

Amino acid residues His and Cys of the NAD-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha H16 were chemically modified with specific reagents. The modification of His residues of the nonactivated hydrogenase resulted in decrease in both hydrogenase and diaphorase activities of the enzyme. Activation of NADH hydrogenase under anaerobic conditions led to the modification of additional His residue (or residues) significant only for the hydrogenase activity. The rate of decrease in the diaphorase activity was unchanged. The modification of thiol groups of the nonactivated enzyme did not affect the activity of the hydrogenase. The effect of thiol-modifying agents on the activated hydrogenase was accompanied by inactivation of both diaphorase and hydrogenase activities. The modification degree and changes in the corresponding catalytic activities depended on conditions of the enzyme activation. Data on the modification of cysteine and histidine residues of the hydrogenase suggested that the enzyme activation should be associated with significant conformational changes in the protein globule.
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PMID:Chemical modification of catalytically essential functional groups of NAD-dependent hydrogenase from Ralstonia eutropha H16. 1460 42

The enzymes pyruvate decarboxylase (E1) and dihydrolipoyl dehydrogenase (E3) bind tightly but in a mutually exclusive manner to the peripheral subunit-binding domain (PSBD) of dihydrolipoyl acetyltransferase in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The use of directed mutagenesis, surface plasmon resonance detection and isothermal titration microcalorimetry revealed that several positively charged residues of the PSBD, most notably Arg135, play an important part in the interaction with both E1 and E3, whereas Met131 makes a significant contribution to the binding of E1 only. This indicates that the binding sites for E1 and E3 on the PSBD are overlapping but probably significantly different, and that additional hydrophobic interactions may be involved in binding E1 compared with E3. Arg135 of the PSBD was also replaced with cysteine (R135C), which was then modified chemically by alkylation with increasingly large aliphatic groups (R135C -methyl, -ethyl, -propyl and -butyl). The pattern of changes in the values of DeltaG degrees, DeltaH degrees and TDeltaS degrees that were found to accompany the interaction with the variant PSBDs differed between E1 and E3 despite the similarities in the free energies of their binding to the wild-type. The importance of a positive charge on the side-chain at position 135 for the interaction of the PSBD with E3 and E1 was apparent, although lysine was found to be an imperfect substitute for arginine. The results offer further evidence of entropy-enthalpy compensation ('thermodynamic homeostasis') - a feature of systems involving a multiplicity of weak interactions.
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PMID:Interactions of the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. 1462 77

The biological effects of nitric oxide (NO) are in significant part mediated through S-nitrosylation of cysteine thiol. Work on model thiol substrates has raised the idea that molecular oxygen (O(2)) is required for S-nitrosylation by NO; however, the relevance of this mechanism at the low physiological pO(2) of tissues is unclear. Here we have used a proteomic approach to study S-nitrosylation reactions in situ. We identify endogenously S-nitrosylated proteins in subcellular organelles, including dihydrolipoamide dehydrogenase and catalase, and show that these, as well as hydroxymethylglutaryl-CoA synthase and sarcosine dehydrogenase (SarDH), are S-nitrosylated by NO under strictly anaerobic conditions. S-Nitrosylation of SarDH by NO is best rationalized by a novel mechanism involving the covalently bound flavin of the enzyme. We also identify a set of mitochondrial proteins that can be S-nitrosylated through multiple reaction channels, including anaerobic/oxidative, NO/O(2), and GSNO-mediated transnitrosation. Finally, we demonstrate that steady state levels of S-nitrosylation are higher in mitochondrial extracts than the intact organelles, suggesting the importance of denitrosylation reactions. Collectively, our results provide new insight into the determinants of S-nitrosothiol levels in subcellular compartments.
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PMID:New insights into protein S-nitrosylation. Mitochondria as a model system. 1506 80

The flavoprotein disulfide reductases represent a family of enzymes that show high sequence and structural homology. They catalyze the pyridine-nucleotide-dependent reduction of a variety of substrates, including disulfide-bonded substrates (lipoamide dehydrogenase, glutathione reductase and functional homologues, thioredoxin reductase, and alkylhydroperoxide reductase), mercuric ion (mercuric ion reductase), hydrogen peroxide (NADH peroxidase), molecular oxygen (NADH oxidase), and the reductive cleavage of a carbonyl-activated carbon-sulfur bond followed by carboxylation (2-ketopropyl-coenzyme-M carboxylase?oxidoreductase). They use at least one nonflavin redox center to transfer electrons from reduced pyridine nucleotide to their substrate through flavin adenine dinucleotide. The nature of the nonflavin redox center located adjacent to the flavin varies and three types have been identified: an enzymic disulfide (most commonly), an enzymic cysteine sulfenic acid (NADH peroxidase and NADH oxidase), and a mixed Cys-S-S-CoA disulfide (coenzyme A disulfide reductase). Selection of the particular nonflavin redox center and utilization of a second, or even a third, nonflavin redox center in some cases presumably represents the most efficient strategy for reduction of the individual substrate.
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PMID:Flavoprotein disulfide reductases: advances in chemistry and function. 1521 Mar 29

The lpdA (Rv3303c) gene from Mycobacterium tuberculosis encoding a new member of the flavoprotein disulfide reductases was expressed in Escherichia coli, and the recombinant LpdA protein was purified to homogeneity. LpdA is a homotetramer and co-purifies with one molecule of tightly but noncovalently bound FAD and NADP+ per monomer. Although annotated as a probable lipoamide dehydrogenase in M. tuberculosis, LpdA cannot catalyze reduction of lipoyl substrates, because it lacks one of two cysteine residues involved in dithiol-disulfide interchange with lipoyl substrates and a His-Glu pair involved in general acid catalysis. The crystal structure of LpdA was solved by multiple isomorphous replacement with anomalous scattering, which confirmed the absence of these catalytic residues from the active site. Although LpdA cannot catalyze reduction of disulfide-bonded substrates, it catalyzes the NAD(P)H-dependent reduction of alternative electron acceptors such as 2,6-dimethyl-1,4-benzoquinone and 5-hydroxy-1,4-naphthaquinone. Significant primary deuterium kinetic isotope effects were observed with [4S-2H]NADH establishing that the enzyme promotes transfer of the C4-proS hydride of NADH. The absence of an isotope effect with [4S-2H]NADPH, the low Km value of 0.5 microm for NADPH, and the potent inhibition of the NADH-dependent reduction of 2,6-dimethyl-1,4-benzoquinone by NADP+ (Ki approximately 6 nm) and 2'-phospho-ADP-ribose (Ki approximately 800 nm), demonstrate the high affinity of LpdA for 2'-phosphorylated nucleotides and that the physiological substrate/product pair is NADPH/NADP+ rather than NADH/NAD+. Modeling of NADP+ in the active site revealed that LpdA achieves the high specificity for NADP+ through interactions involving the 2'-phosphate of NADP+ and amino acid residues that are different from those in glutathione reductase.
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PMID:Characterization of a new member of the flavoprotein disulfide reductase family of enzymes from Mycobacterium tuberculosis. 1545 92


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