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)

1. Kinetic studies of lipoamide dehydrogenase and its modified enzymes catalyzing lipoamide oxidoreduction and ancillary reactions at various pH are compared. 2. The asymptotic kinetics of lipoamide oxidoreductions switch between the ping pong and ordered mechanisms by varying pH of the reactions. 3. pH-rate profiles of these reactions are bell-shaped suggesting the participation of 2 ionizable residues with pK values of 6.6 +/- 0.5 and above 8 respectively. 4. The unusually high pK value for the catalytic site histidine is attributed to its involvement in an ion-pair formation. 5. In the absence of the catalytic site histidine, the pH-rate profile for the lipoamide reduction of the photooxidized enzyme is no longer bell-shaped but it is similar to those of the transhydrogenation and NADH-oxidation of the native enzyme. 6. This implies the participation of a low-pK protonated group in these reactions.
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PMID:pH dependent kinetic studies of lipoamide dehydrogenase catalysis. 145 16

Lipid peroxidation of rat erythrocyte membranes was induced by lipoamide dehydrogenase (LADH) (EC 1.8.1.4) in the presence of ADP-Fe3+. Superoxide dismutase (SOD) (EC 1.15.1.1) strongly inhibited the peroxidation reaction but catalase did not. Hydroxyl radical scavengers, mannitol and dimethylsulfoxide did not inhibit the lipid peroxidation. These results indicated that the lipid peroxidation was a superoxide (O2-)-dependent reaction, but the hydroxyl radical was not involved. ADP-Fe3+, in the presence of LADH, was reduced more rapidly under aerobic than anaerobic conditions and SOD under aerobic conditions strongly inhibited the iron reduction, indicating that O2- plays a predominant role in iron reduction. Hydrogen peroxide enhanced O2- generation by LADH, but the peroxidation reaction was not affected. In the presence of lipoamide, lipid peroxidation was also induced but the reactions were not inhibited by SOD. Evidently, the lipid peroxidation induced in the presence of lipoamide was O2(-)-independent. Dihydrolipoamide may be involved in the peroxidation reaction.
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PMID:Lipid peroxidation of erythrocyte membrane induced by lipoamide dehydrogenase in the presence of ADP-Fe3+. 145 54

In order to elucidate the mechanism of the biological activation of nitrofurans, the interaction of these compounds with lipoamide dehydrogenase (LipDH)** was investigated. LipDH catalysed one-electron reduction of several nitrofuran derivatives. The reaction could be demonstrated spectroscopically and was enhanced by cadmium, arsenite and anaerobiosis. The role of flavin in the nitroreductase activity was supported by (a) the nitrofuran effect on the spectral properties of anaerobic, arsenite-inhibited, NADH-reduced LipDH; (b) FAD catalytic activity in a NADH-nitrofuran model system; and (c) the nitroreductase activity of LipDH monomer. Two-electron nitrofuran reduction to less oxidized products was inhibited by cadmium, arsenite and NAD+. The possible role of reactive nitrosofuran derivatives as intermediates of the nitrofuran reduction sequence was supported by the LipDH capability for catalysing 2-nitroso-1-naphthol redox-cycling. The nitroso naphthol reduction was inhibited by cadmium and arsenite, like the two-electron nitrofuran reduction.
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PMID:Reduction of nitrofuran compounds by heart lipoamide dehydrogenase: role of flavin and the reactive disulfide groups. 145 54

A dihydrolipoamide dehydrogenase (dihydrolipoamide: NAD+ oxidoreductase, EC 1.8.1.4) (DLD) has been found in the soluble fraction of cells of both unicellular (Synechococcus sp. strain P.C.C. 6301) and filamentous (Calothrix sp. strain P.C.C. 7601 and Anabaena sp. strain P.C.C. 7119) cyanobacteria. DLD from Anabaena sp. was purified 3000-fold to electrophoretic homogeneity. The purified enzyme exhibited a specific activity of 190 units/mg and was characterized as a dimeric FAD-containing protein with a native molecular mass of 104 kDa, a Stokes' radius of 4.28 nm and a very acidic pI value of about 3.7. As is the case with the same enzyme from other sources, cyanobacterial DLD showed specificity for NADH and lipoamide, or lipoic acid, as substrates. Nevertheless, the strong acidic character of the Anabaena DLD is a distinctive feature with respect to the same enzyme from other organisms. The presence of essential thiol groups was suggested by the inactivation produced by thiol-group-reactive reagents and heavy-metal ions, with lipoamide, but not NAD+, behaving as a protective agent. The function and physiological significance of Anabaena DLD are discussed in relation to the fact that 2-oxoacid dehydrogenase complexes have not been detected so far in filamentous cyanobacteria. Glycine decarboxylase activity, which might be involved in photorespiratory metabolism, has been found, however, in cell extracts of Anabaena sp. strain P.C.C. 7119 as the present study demonstrates.
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PMID:Purification, characterization and function of dihydrolipoamide dehydrogenase from the cyanobacterium Anabaena sp. strain P.C.C. 7119. 147 97

Saccharomyces cerevisiae possesses 2-oxoacid dehydrogenase (EC 1.2.4.4) similar to that found in mammalian cells. The activity is readily detected in cells which have been cultured in a minimal medium containing a branched-chain amino acid. Mutants defective in lipoamide dehydrogenase also lack 2-oxoacid dehydrogenase and are thus unable to catabolize branched-chain amino acids: 2-oxoacids accumulate in the cultures of these cells. The 2-oxoacid dehydrogenase activity is distinct from both 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase, because it could not be detected in assay conditions which permitted the measurement of 2-oxoglutarate dehydrogenase and vice versa. In addition, a strain lacking 2-oxoglutarate dehydrogenase (kgd1::URA3) retained 2-oxoacid dehydrogenase as did a mutant specifically lacking pyruvate dehydrogenase (pda1::Tn5ble). In complex media the specific activity of this enzyme is highest in YEP (yeast extract-peptone)-glycerol and lowest in YEP-acetate and YEP-fructose. 2-Oxoacid dehydrogenase could not be detected in cells which had been transferred to sporulation medium. These results suggest that in S. cerevisiae the catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase, not via the 'Ehrlich Pathway'.
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PMID:The catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase in Saccharomyces cerevisiae. 147 41

An arsenical resistant cloned line of Trypanosoma brucei brucei was derived from a parent sensitive clone by repeated selection in vivo with the pentavalent melaminophenyl arsenical, sodium melarsen. The melarsen-resistant line was tested in vivo in mice against a range of trypanocidal compounds and found to be cross-resistant to the trivalent arsenicals, melarsen oxide, melarsoprol and trimelarsen (33, 67 and 122-fold, respectively). A similar pattern of cross-resistance was found in vitro using a spectrophotometric lysis assay (greater than 200-fold resistance to melarsen oxide and greater than 20-fold resistance to both trimelarsen and melarsoprol). Both lines were equally sensitive to lysis by the lipophilic analogue phenylarsine oxide in vitro, suggesting that the melamine moiety is involved in the resistance mechanism. Although trypanothione has been reported to be the primary target for trivalent arsenical drugs [1], levels of trypanothione and glutathione were not significantly different between the resistant and sensitive lines. Statistically significant differences were found in the levels of trypanothione reductase (50% lower in the resistant clone) and dihydrolipoamide dehydrogenase (38% higher in the resistant clone). However, the Km for trypanothione disulphide, the Ki for the competitive inhibitor Mel T (the melarsen oxide adduct with trypanothione) and the pseudo-first order inactivation rates with melarsen oxide were the same for trypanothione reductase purified from both clones. The melarsen-resistant line also showed varying degrees of cross-resistance to the diamidines: stilbamidine (38-fold), berenil (31.5-fold), propamidine (5.7-fold) and pentamidine (1.5-fold). Cross-resistance correlates with the maximum interatomic distance between the amidine groups of these drugs and suggests that the diamidines and melaminophenyl arsenicals are recognised by the same transport system.
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PMID:Characterisation of melarsen-resistant Trypanosoma brucei brucei with respect to cross-resistance to other drugs and trypanothione metabolism. 150 41

D,L-dihydrolipoamide and D,L-dihydrolipoic acid react to form stable complexes with melarsen oxide with association constants of 5.47 x 10(9) and 4.51 x 10(9) M-1, respectively. These complexes possess 6-membered cyclic dithioarsenite rings which are 10-fold less stable than the 5-membered rings found in the trypanocidal drugs melarsoprol and trimelarsen, but 500-fold more stable than the 25-membered macrocyclic ring formed between melarsen oxide and dihydrotrypanothione. L-Lipoic acid concentrations in arsenical sensitive and resistant cloned lines of Trypanosoma brucei brucei have been determined by bioassay using a mutant of Escherichia coli auxotrophic for lipoate. The arsenical resistant strain was found to contain significantly less lipoic acid than the sensitive strain (19.2 +/- 4.3 and 9.7 +/- 2.9 pmol (10(8) cells)-1, respectively). The activity of the plasma membrane-associated dihydrolipoamide dehydrogenase was found to be slightly, but significantly increased in the arsenical resistant strain (34.7 +/- 1.4 and 47.8 +/- 3.7 mU mg-1, respectively). However, the Km for dihydrolipoamide and the inactivation kinetics with melarsen oxide were not significantly different between these strains. Estimates of the ratio of substrate to enzyme are of the order of 12:1 and 6:1 for arsenical sensitive and resistant strains, respectively, suggesting that these components are likely to be intimately associated with each other in the plasma membrane. These findings implicate lipoic acid, but not dihydrolipoamide dehydrogenase, in resistance to arsenical drugs, either through the mechanism of uptake or as the final target of these drugs.
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PMID:The interaction of arsenical drugs with dihydrolipoamide and dihydrolipoamide dehydrogenase from arsenical resistant and sensitive strains of Trypanosoma brucei brucei. 150 42

Sequences located in the N-terminal region of the high M(r) 2-oxoglutarate dehydrogenase (E1) enzyme of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex (OGDC) exhibit significant similarity with corresponding sequences from the lipoyl domains of the dihydrolipoamide acetyltransferase (E2) and protein X components of eukaryotic pyruvate dehydrogenase complexes (PDCs). Two additional features of this region of E1 resemble lipoyl domains: (i) it is readily released by trypsin, generating a small N-terminal peptide with an apparent M(r) value of 10,000 and a large stable 100,000 M(r) fragment (E1') and (ii) it is highly immunogenic, inducing the bulk of the antibody response to intact E1. This 'lipoyl-like' domain lacks a functional lipoamide group. Selective but extensive degradation of E1 with proteinase Arg C or specific conversion of E1 to E1' with trypsin both cause loss of overall OGDC function although the E1' fragment retains full catalytic activity. Removal of this small N-terminal peptide promotes the dissociation of dihydrolipoamide dehydrogenase (E3) from the E2 core assembly and also affects the stability of E1 interaction. Thus, structural roles which are mediated by a specific gene product, protein X in PDC and possibly also the E2 subunit, are performed by similar structural elements located on the E1 enzyme of the OGDC.
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PMID:Sequences directing dihydrolipoamide dehydrogenase (E3) binding are located on the 2-oxoglutarate dehydrogenase (E1) component of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex. 150 15

L-protein is the dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a lambda gt11 pea library encoding the complete L-protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the glycine-cleavage system recognized proteins with an Mr of about 60000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected. Second, Northern-blot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H-protein (a specific protein component of the glycine-cleavage system) is known to be expressed primarily in leaves. Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.
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PMID:Isolation, characterization, and sequence analysis of a cDNA clone encoding L-protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea-leaf mitochondria. 154 Dec 97

A well-defined set of isogenic yeast strains has been constructed whereby each strain contains a different LPD::lacZ gene fusion integrated at the ura3 locus. These LPD::lacZ fusions differ in the amount of the LPD1 gene (encoding lipoamide dehydrogenase) that is fused to the lacZ reporter. Comparison of the beta-galactosidase activities of each strain during growth on glucose or ethanol revealed that some part of the LPD1 coding region between +13 and +700 is involved in activating gene expression in a carbon source-dependent manner. This activation occurs at the mRNA level, and is not mediated by changes in mRNA stability. Therefore, the LPD1 gene appears to contain a transcriptional enhancer that lies 3' to the transcriptional start site, and which responds to carbon source.
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PMID:A 3' transcriptional enhancer within the coding sequence of a yeast gene encoding the common subunit of two multi-enzyme complexes. 154 8


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