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)

Thioredoxin from Escherichia coli was shown to catalyze the reduction of insulin disulfides by dithiothreitol. A quantitative assay was developed which measures the rate of insulin reduction spectrophotometrically at 650 nm as turbidity formation from the precipitation of the free insulin B chain. Thioredoxin, at 5 microM concentration, accelerated the reaction between 0.130 mM insulin and 1.0 mM dithiothreitol at pH 7 around 20-fold. The pH optimum of the reaction was 7.5. Thioredoxins from E. coli and calf liver showed similar specific activities. Stopped flow fluorescence measurements of the rate of reduction of thioredoxin-S2 by dithiothreitol showed a second order rate constant of 1647 M-1 s-1 at pH 7.2. This is between 10(2) to 10(3) times larger than the reaction between insulin or linear model disulfides and dithiothreitol. It is consistent with a ping-pong mechanism of thioredoxin catalysis since reduced thioredoxin is known to react very fast with insulin. Thioredoxin also catalyzed lipoamide-dependent reduction of the insulin disulfides in a coupled system with NADH, lipoamide, and lipoamide dehydrogenase. The fast spontaneous reaction between dihydrolipoamide and thioredoxin-S2 provides a mechanism for NADH or pyruvate-dependent disulfide reduction. The implication of the dithiol-disulfide oxidoreductase activity of thioredoxin for the regulation of enzyme activities by thiol oxidation-reduction control is discussed.
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PMID:Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. 38 88

Purification of protein PA of the glycine reductase complex from Eubacterium acidaminophilum and Clostridium litorale [corrected] was monitored by a new spectrophotometric assay. The procedure depended on a specific two- to threefold stimulation of a dihydrolipoamide dehydrogenase activity that is elicited by the interaction of a thioredoxin reductase-like flavoprotein and thioredoxin from both organisms. Protein PA isolated from E. acidaminophilum by 75Se labeling and monitoring of the dithioerythritol-dependent glycine reductase activity was identical in its biochemical, structural, and immunological properties to the protein isolated by using the stimulation assay. Proteins PA from both organisms were glycoproteins of Mr about 18,500 and exhibited very similar N-terminal amino acid sequences. Depletion of thioredoxin from crude extracts of E. acidaminophilum totally diminished the NADPH-dependent but not the dithioerythritol-dependent glycine reduction. The former activity could be fully restored by adding thioredoxin. Antibodies raised against the thioredoxin reductase-like flavoprotein or thioredoxin inhibited to a high extent NADPH-dependent but not dithioerythritol-dependent glycine reductase activity. These results indicate the involvement of the thioredoxin system in the electron flow from reduced pyridine nucleotides to glycine reductase.
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PMID:Interaction of selenoprotein PA and the thioredoxin system, components of the NADPH-dependent reduction of glycine in Eubacterium acidaminophilum and Clostridium litorale [corrected]. 191 32

The glycine-utilizing bacterium Clostridium litoralis contained two enzyme systems for oxidizing dihydrolipoamide. The first one was found to be a genuine dihydrolipoamide dehydrogenase, present only in low amounts. This enzyme had the typical dimeric structure with a subunit molecular mass of about 53 kDa; however, it reacted with both NADP (Km 0.11 mM) and NAD (Km 0.5 mM). The reduction of pyridine nucleotides by dihydrolipoamide was the strongly preferred reaction. A second dihydrolipoamide-oxidizing enzyme system consisted of the interaction of two proteins, the previously described NADP(H)-dependent electron-transferring flavoprotein (D. Dietrichs, M. Meyer, B. Schmidt, and J. R. Andreesen, J. Bacteriol. 172:2088-2095, 1990) and a thioredoxin. This enzyme system was responsible for most of the dihydrolipoamide dehydrogenase activity in cell extracts. The thioredoxin did not bind to DEAE, was heat stable, and had a molecular mass of about 15 kDa. N-terminal amino acid analysis of the first 38 amino acid residues resulted in 38% homology to Escherichia coli thioredoxin and about 76% homology to a corresponding protein isolated from the physiologically close related Eubacterium acidaminophilum. The protein of the latter organism had a molecular mass of about 14 kDa and stimulated the low dihydrolipoamide dehydrogenase activity of the corresponding flavoprotein. By this interaction with NADPH-dependent flavoproteins, a new assay system for thioredoxin was established. A function of thioredoxin in glycine metabolism of some anaerobic bacteria is proposed.
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PMID:Thioredoxin elicits a new dihydrolipoamide dehydrogenase activity by interaction with the electron-transferring flavoprotein in Clostridium litoralis and Eubacterium acidaminophilum. 199 93

The thioredoxin peptide Trp-Cys-Gly-Pro-Cys-Lys, which contains the redox active dithiol, was found to be reduced by lipoamide in a coupled reaction with lipoamide dehydrogenase and NADH. The reduced peptide in turn was shown to reduce insulin, oxidized lens protein and glyceraldehyde-3-phosphate dehydrogenase. While the peptide is not as effective a catalyst for utilizing pyridine nucleotides to reduce protein disulfides as thioredoxin, it offers a system which may be developed to provide more efficient disulfide reduction. This is particularly relevant since no thioredoxin peptides have been found to be active with thioredoxin reductase.
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PMID:Thioredoxin fragment 31-36 is reduced by dihydrolipoamide and reduces oxidized protein. 312 52

The flavoprotein thioredoxin reductase catalyzes the reduction of the small redox protein thioredoxin by NADPH. Thioredoxin reductase contains a redox active disulfide and is a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes that includes lipoamide dehydrogenase, glutathione reductase, trypanothione reductase, mercuric reductase, and NADH peroxidase. The structure of thioredoxin reductase has recently been determined from X-ray crystallographic data. In this paper, we attempt to correlate the structure with a considerable body of mechanistic data and to arrive at a mechanism consistent with both. The path of reducing equivalents in catalysis by glutathione reductase and lipoamide dehydrogenase is clear. To envisage the path of reducing equivalents in catalysis by thioredoxin reductase, a conformational change is required in which the NADPH domain rotates relative to the FAD domain. The rotation moves the nascent dithiol from its observed position adjacent to the re surface of the flavin ring system toward the protein surface for dithiol-disulfide interchange with the protein substrate thioredoxin and moves the nicotinamide ring of NADPH adjacent to the flavin ring for efficient hydride transfer. Reverse rotation allows reduction of the redox active disulfide by the reduced flavin. This requires that the enzyme pass through a ternary complex; the kinetic evidence for such a complex is discussed.
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PMID:Mechanism and structure of thioredoxin reductase from Escherichia coli. 755 16

The thioredoxin-catalyzed insulin reduction by dihydrolipoate was applied to study the 2-oxoacid: lipoate oxidoreductase activity of 2-oxoacid dehydrogenase complexes. The enzymatic and non-enzymatic mechanisms of the transfer of reducing equivalents from the complexes to free lipoic acid (alpha-lipoic acid, 6,8-thiooctic acid) were distinguished using the high stereoselectivity of the complex enzymes to the R-enantiomer of lipoate. Unlike these enzymes, thioredoxin from E. coli exhibited no stereoselectivity upon reduction with chemically obtained dihydrolipoate. However, coupled to the dihydrolipoate production by the dehydrogenase complexes, the process was essentially sensitive both to the enantiomer used and the dihydrolipoyl dehydrogenase activity of the complexes. These results indicated the involvement of the third complex component, dihydrolipoyl dehydrogenase, in the 2-oxoacid-dependent dihydrolipoate formation. The implication of the investigated reaction for a connection between thioredoxin and the 2-oxoacid dehydrogenase complexes in the mitochondrial metabolism are discussed.
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PMID:Using lipoate enantiomers and thioredoxin to study the mechanism of the 2-oxoacid-dependent dihydrolipoate production by the 2-oxoacid dehydrogenase complexes. 767 20

The genes encoding thioredoxin reductase (trxB), thioredoxin (trxA), protein PA of glycine reductase (grdA) and the first 23 amino acids of the large subunit of protein PC of glycine reductase (grdC) belonging to the reductive deamination systems present in Eubacterium acidaminophilum were cloned and sequenced. The proteins were products of closely linked genes with 314 codons (thioredoxin reductase), 110 codons (thioredoxin), and 158 codons (protein PA). The protein previously called 'atypically small lipoamide dehydrogenase' or 'electron transferring flavoprotein' could now conclusively be identified as a thioredoxin reductase (subunit mass of 34781 Da) by the alignment with the enzyme of Escherichia coli showing the same typical order of the corresponding domains. The thioredoxin (molecular mass of 11742 Da) deviated considerably from the known consensus sequence, even in the most strongly conserved redox-active segment WCGPC that was now GCVPC. The selenocysteine of protein PA (molecular mass of 16609 Da) was encoded by TGA. The protein was highly similar to those of Clostridium purinolyticum and Clostridium sticklandii involved in glycine reductase. Thioredoxin reductase and thioredoxin of E. acidaminophilum could be successfully expressed in E. coli.
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PMID:Components of glycine reductase from Eubacterium acidaminophilum. Cloning, sequencing and identification of the genes for thioredoxin reductase, thioredoxin and selenoprotein PA. 822 22

Selenium is a potent chemopreventive agent; however, the mechanisms for its chemopreventive activities remain elusive. Selenium binds to several proteins, some of which require selenium for functional activity. In this study, two 58kDa selenium-labeled proteins were identified in mouse kidney using a 75Se labeling method. The proteins were partially purified using Sephadex G.150 gel filtration, DEAE-Sephadex A-50 ion-exchange chromatography and one- / two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-/2D-SDS-PAGE). The two proteins migrated at 58kDa on 2D-SDS-PAGE and differed only slightly in their pI values; i.e., 6.2 and 6.6, respectively. The polyclonal antibodies raised in rabbits against the 58kDa proteins electro-eluted from the 1D-SDS-PAGE of the DEAE purified fraction, recognized both protein spots on 2D-SDS-PAGE gel. The in situ enzymatic digestion of the two proteins separated in 2D-SDS-PAGE gels, followed by microsequencing of the peptides, resulted in the identification of these two proteins as related to human lipoamide dehydrogenase and thiol: protein disulfide oxidoreductase (TPDO). In common, both these proteins have a bis (cysteinyl) sequence motif cys-X-X-cys (for lipoamide dehydrogenase it is cys-X-X-X-X-cys) which is also an integral part of several other proteins such as thioredoxin, protein disulfide isomerase, endoplasmic reticulum protein (ERp72), selenoprotein W, 56kDa acetaminophen binding protein and formate dehydrogenase. This sequence motif acts as an active redox center for majority of the proteins mentioned above, that may be controlling the oxidation/reduction of proteins in vivo. How and why selenium is binding to proteins with this common sequence motif needs further investigation.
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PMID:Isolation and identification of selenium-labeled proteins in the mouse kidney. 871 83

The thioredoxin system, composed of the pyridine nucleotide-disulfide oxidoreductase thioredoxin reductase, the small peptide thioredoxin, and NADPH as a reducing cofactor, is one of the major thiol-reducing systems of the cell. Recent studies revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, called large thioredoxin reductases. The large thioredoxin reductases are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. The putative essential amino acids at the catalytic center of large thioredoxin reductase from P. falciparum were determined by using site-directed mutagenesis techniques. To analyze the putative active site cysteines (Cys88 and Cys93) three mutant proteins were constructed substituting alanine or serine residues for cysteine residues. Further, to evaluate the function of His509 as a putative proton donor/acceptor of large thioredoxin reductase this residue was replaced by either glutamine or alanine. All mutants were expressed in the E. coli system and characterized. Steady state kinetic analysis revealed that the replacement of Cys88 by either alanine or serine and Cys93 by alanine resulted in a total loss of enzymatic activity. These results clearly identify Cys88 and Cys93 as the active site thiols of large thioredoxin reductase. The replacement of His509 by glutamine yielded in a 95% loss of thioredoxin reductase activity; replacement by alanine provoked a loss of 97% of enzymatic activity. These results identify His509 as active site base, but imply that its function can be substituted, although inefficiently, by an alternative proton donor, similar to glutathione reductase. Spectral analysis of wild-type P. falciparum thioredoxin reductase revealed a 550-nm absorption band upon reduction which resembles the EH2 form of glutathione reductase and lipoamide dehydrogenase. This spectral feature, recently also reported for the human placenta protein (Arscott, L. D., Gromer, S., Schirmer, R. H., Becker K., and Williams, C. H., Jr. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3621-3626), further illustrates the similarity between large thioredoxin reductases and glutathione reductases and stresses the profound differences to small E. coli thioredoxin reductase.
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PMID:Identification and characterization of the functional amino acids at the active site of the large thioredoxin reductase from Plasmodium falciparum. 936 22

Thioredoxin reductase (EC 1.6.4.5) is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin. Thioredoxin plays several key roles in maintaining the redox environment of the cell. Like all members of the enzyme family that includes lipoamide dehydrogenase, glutathione reductase and mercuric reductase, thioredoxin reductase contains a redox active disulfide adjacent to the flavin ring. Evolution has produced two forms of thioredoxin reductase, a protein in prokaryotes, archaea and lower eukaryotes having a Mr of 35 000, and a protein in higher eukaryotes having a Mr of 55 000. Reducing equivalents are transferred from the apolar flavin binding site to the protein substrate by distinct mechanisms in the two forms of thioredoxin reductase. In the low Mr enzyme, interconversion between two conformations occurs twice in each catalytic cycle. After reduction of the disulfide by the flavin, the pyridine nucleotide domain must rotate with respect to the flavin domain in order to expose the nascent dithiol for reaction with thioredoxin; this motion repositions the pyridine ring adjacent to the flavin ring. In the high Mr enzyme, a third redox active group shuttles the reducing equivalent from the apolar active site to the protein surface. This group is a second redox active disulfide in thioredoxin reductase from Plasmodium falciparum and a selenenylsulfide in the mammalian enzyme. P. falciparum is the major causative agent of malaria and it is hoped that the chemical difference between the two high Mr forms may be exploited for drug design.
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PMID:Thioredoxin reductase two modes of catalysis have evolved. 1101 62


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