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)

The molar ratio of the component enzymes of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was found to be 1.8:1.7:1[pyruvate decarboxylase (E1):dihydrolipoyl transacetylase (E2):dihydrolipoyl dehydrogenase (E3)]. This ratio was determined by measuring the Coomassie blue staining of the constituent enzymes after sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. The above ratio is the average of four separate experiments with two different enzyme preparations. The average molecular weights of the individual enzymes were found to be 96,000, 76,000, and 55,000 for E1, E2, and E3, respectively, by sodium dodecyl sulfate and sodium dodecyl sulfate/8 M urea polyacrylamide gel electrophoresis and by column chromatography in 6 M guanidine . HCl. The molecular weight of E2 was reduced to 33,000-36,000 after extensive reduction and alkylation with iodoacetamide. The molecular weights of the complex, E1, and E3 were found to be 4,800,000, 182,000, and 104,000, respectively, with low-angle laser light scattering. Both E1 and E3 are dimeric under the conditions employed. If octahedral symmetry is assumed for the E2 core, a polypeptide chain ratio of 24:24:12 (E1:E2:E3) is in good agreement with the measured molar ratio of component enzymes and the molecular weight of the pyruvate dehydrogenase complex.
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PMID:Subunit stoichiometry and molecular weight of the pyruvate dehydrogenase multienzyme complex from Escherichia coli. 38 35

1. The effect of guanidine hydrochloride (GuHCl) on pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3.] was investigated by means of enzymatic activity and optical measurements (CD, absorption, and fluorescence spectra). The activity of the enzyme decreased on increasing the concentration of GuHCl and the enzyme was completely inactivated in 2.0 M GuHCl. 2. The contents of alpha-helix, beta, and unordered forms in lipoamide dehydrogenase were estimated to be 34, 14, and 52%, respectively. On increasing the concentration of GuHCl, the content of alpha-helix in lipoamide dehydrogenase decreased, whereas the content of the beta form hardly changed. 3. The native lipoamide dehydrogenase showed absorption, CD, and fluorescence spectra characteristic of bound FAD in the visible region, suggesting hydrophobic interaction between the protein moiety and FAD chromophore. The absorption, CD, and fluorescence spectra of the enzyme in 2.0 M GuHCl were similar to those of free FAD in the buffer, suggesting the release of FAD from the protein moiety. 4. The protein fluorescence spectrum of lipoamide dehydrogenase had a maximum at 350 nm blue-shifted by 8 nm from that of tryptophan in aqueous solution. The maximum of the enzyme in 2.0 M GuHCl was red-shifted to 357 nm. This suggests exposure of tryptophan residues to a polar environment. The maximum, 352nm, of the apoenzyme shifted to 350 nm on addition of FAD. These results show that the conformation in the microenvironment of some tryptophan residues in lipoamide dehydrogenase is affected by the dissociation-association of FAD. 5. The contents of alpha-helix, beta, and unordered forms in the apoenzyme were estimated to be 35, 8, and 57%, respectively. These values are similar to those of the native holoenzyme. The alpha-helical structure in the apoenzyme molecule was more sensitive to GuHCl than that in the holoenzyme. FAD and two hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4 benzolamido-4'-aminostilbene-2,2'-disulfonate (MBAS), which can bind to the apoenzyme, stabilized the alpha-helical structure in the apoenzyme molecule.
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PMID:Effect of guanidine hydrochloride on the holo- and apo-enzymes of pig heart lipoamide dehydrogenase. 93 80

The interaction of hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4-benzoylamido-4'-aminostilbene-2, 2'-disulfonate (MBAS), with pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3] was investigated. When ANS or MBAS was mixed with the apoenzyme of lipoamide dehydrogenase, the fluorescence quantum yield, of each dye was enhancedd markedly and the emission maxima concurrently shifted to the blue. The quantum yield, 0.038, of ANS bound to the apoenzyme, calculated from the corrected emission spectrum, was eight times higher than that in buffer solution, and the value, 0.0090, for bound MBAS was eighteen times higher than that in buffer solution. Moreover, the absortion bands of both ANS and MBAS shifted to the red upon binding with the apoenzyme. A general feature of the absorption spectra of these dyes observed on changing the solvent from polar to apolar was a red shift of the absorption bands. These results indicate that ANS or MBAS bound to the apoenzyme of lipoamide dehydrogenase is situated in a hydrophobic region of the apoenzyme molecule. It was found that 2 moles of each dye was bound per mole of the apoenzyme, which contains two polypeptide chains. The dissociation constants for the ANS- and MBAS-apoenzyme complexes were estimated to be 1.03X10(-5) and 1.54X10(-5) M, respectively. The enhanced fluorescence of both dyes bound to the apoenzyme decreased linearly upon adding FAD and disappeared at about 2 moles of FAD per mole of the apoenzyme. This suggests that both ANS and MBAS were displaced from their binding sites on the apoenzyme by FAD. The protein fluorescence spectrum of the apoenzyme had a maximum at 352 nm, which was blue-shifted by 6 nm from that of tryptophan in the buffer. Upon binding ANS or MBAS, the maximum of the protein fluorescence of the apoenzyme returned to 350 nm for the holoenzyme, and the fluorescence intensity decreased. Thus, the conformation around some tryptophan residues was affected by the binding of the dyes. When guanidine hydrochloride (GuHCl) was added to the ANS-apoenzyme complex solution, the enhanced fluorescence due to the bound ANS decreased and the emission maximum concurrently shifted to the red. Further, the maximum of the protein fluorescence of the apoenzyme shifted to the red, indicating the exposure of some tryptophan residues buried in an apolar region of the apoenzyme. Thus the binding of ANS to the apoenzyme was inhibited by protein denaturation due to GuHCL. In contrast, the holoenzyme of lipoamide dehydrogenase did not bind ANS or MBAS at all.
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PMID:Interaction of hydrophobic probes with the apoenzyme of pig heart lipoamide dehydrogenase. 95 45

The conformational stability of holo-lipoamide and apo-lipoamide dehydrogenase from Azotobacter vinelandii was studied by thermoinactivation, unfolding and limited proteolysis. The oxidized holoenzyme is thermostable, showing a melting temperature, tm = 80 degrees C. The thermal stability of the holoenzyme drastically decreases upon reduction. Unlike the oxidized and lipoamide two-electron reduced enzyme species, the NADH four-electron reduced enzyme is highly sensitive to unfolding by urea. Loss of energy transfer from Trp199 to flavin reflects the unfolding of the oxidized holoenzyme by guanidine hydrochloride. Unfolding of the monomeric apoenzyme is a rapid fully reversible process, following a simple two-state mechanism. The oxidized and two-electron reduced holoenzyme are resistant to limited proteolysis by trypsin and endoproteinase Glu-C. Upon cleavage of the apoenzyme or four-electron reduced holoenzyme by both proteases, large peptide fragments (molecular mass greater than 40 kDa) are transiently produced. Sequence studies show that limited trypsinolysis of the NADH-reduced enzyme starts mainly at the C-terminus of Arg391. In the apoenzyme, limited proteolysis by endoproteinase Glu-C starts from the C-terminus at the carboxyl ends of Glu459 and/or Glu435. From crystallographic data it is deduced that the susceptible amino acid peptide bonds are situated near the subunit interface. Thus, these bonds are inaccessible to the proteases in the dimeric enzyme and become accessible after monomerization. It is concluded that reduction of lipoamide dehydrogenase to the four-electron reduced state(s) is accompanied by conformational changes promoting subunit dissociation.
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PMID:The conformational stability of the redox states of lipoamide dehydrogenase from Azotobacter vinelandii. 176 65

After limited proteolysis of the dihydrolipoyl transacetylase component (E2) of Azotobacter vinelandii pyruvate dehydrogenase complex (PDC), a C-terminal domain was obtained which retained the transacetylase active site and the quaternary structure of E2 but had lost the lipoyl-containing N-terminal part of the chain and the binding sites for the peripheral components, pyruvate dehydrogenase and lipoamide dehydrogenase. The C-terminus of this domain was determined by treatment with carboxypeptidase Y and shown to be identical with the C-terminus of E2. Together with the previously determined N-terminus and the known amino acid sequence of E2, a molecular mass of 27.5 kDa was calculated. From the molecular mass of the native catalytic domain, 530 kDa, and the symmetry of the cubic structures observed on electron micrographs, a 24-meric structure is concluded instead of the 32-meric structure proposed previously. From the effect of guanidine hydrochloride on the light-scattering of intact E2 it was concluded that dissociation occurs in a two-step reaction resulting in particles with an average mass 1/6 that of the original mass before the N----D transition takes place. Cross-linking experiments with the catalytic domain indicated that the multimeric E2 is built from tetramers and that the tetramers are arranged as a dimer of dimers. A model for the quaternary structure of E2 is given, in which it is assumed that the tetrameric E2 core of PDC is formed from each of the six morphological subunits located at the lateral face of the cube. Binding of peripheral components to a site that interferes with the cubic assembly causes dissociation, resulting in the unique small PDC of A. vinelandii.
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PMID:The quaternary structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. A reconsideration. 291 67

Limited proteolysis with trypsin has been used to study the domain structure of the dihydrolipoyltransacetylase (E2) component of the pyruvate dehydrogenase complex of Azotobacter vinelandii. Two stable end products were obtained and identified as the N-terminal lipoyl domain and the C-terminal catalytic domain. By performing proteolysis of E2, which was covalently attached via its lipoyl groups to an activated thiol-Sepharose matrix, a separation was obtained between the catalytic domain and the covalently attached lipoyl domain. The latter was removed from the column after reduction of the S-S bond and purified by ultrafiltration. The lipoyl domain is monomeric with a mass of 32.6 kDa. It is an elongated structure with f/fo = 1.62. Circulair dichroic studies indicates little secondary structure. The catalytic domain is polymeric with S20.w = 17 S and mass = 530 kDa. It is a compact structure with f/fo = 1.24 and shows 40% of the secondary structure of E2. The cubic structure of the native E2 is retained by this fragment as observed by electron microscopy. Ultracentrifugation in 6 M guanidine hydrochloride in the presence of 2 mM dithiothreitol yields a mass of 15.8 kDa. An N-terminal sequence of 36 amino acids is homologous with residues 370-406 of Escherichia coli E2. The catalytic domain possesses the catalytic site, but in contrast to the E. coli subunit binding domain the pyruvate dehydrogenase (E1) and lipoamide dehydrogenase (E3) binding sites are lost during proteolysis. From comparison with the E. coli E2 sequence a model is presented in which the several functions, such as lipoyl domain, the E3 binding site, the catalytic site, the E2/E2 interaction sites, and the E1 binding site, are indicated.
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PMID:The domain structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. 369 94

A novel procedure was developed for rapid separation of the three component enzymes of pig heart 2-oxoglutarate dehydrogenase complex by high performance liquid chromatography on a gel filtration column. The complex was dissociated and separated into two fractions of the first dihydrolipoamide succinyltransferase and a second yellow fraction within 1 h by chromatography on a preparative TSK-GEL G4000SW column equilibrated with 0.05 M potassium phosphate buffer (pH 7.0) containing 0.7 M guanidine hydrochloride, 0.05% Triton X-100 and 2 mM dithiothreitol at 10 degrees C. The dihydrolipoamide succinyltransferase fraction was further purified by incubation with 0.5% sodium deoxycholate and subsequent ammonium sulfate fractionation. The other two component enzymes, 2-oxoglutarate dehydrogenase and lipoamide dehydrogenase were separated from the second yellow fraction by chromatography on a calcium phosphate gel-cellulose column. The TSK-GEL column permitted very rapid dissociation and separation of the three component enzymes accompanied by good preservation of their activities and high overall yields.
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PMID:Rapid and simple isolation procedure for three component enzymes of pig heart 2-oxoglutarate dehydrogenase complex. 375 90

1. Pyruvate dehydrogenase complex from Saccharomyces cerevisiae is similar in size (s20,w 77 S) and flavin content (1.3--1.4 nmol/mg) to the complexes from mammalian mitochondria. 2. The relative molecular masses of the constituent polypeptide chains, as determined by dodecylsulfate gel electrophoresis at different gel concentrations, were: lipoate acetyltransferase (E2), 58 000; lipoamide dehydrogenase (E3), 56 000; pyruvate dehydrogenase (E1), alpha-subunit, 45 000, and beta-subunit, 35 000. Gel chromatography in the presence of 6 M guanidine . HCl gave a value of 52 000 for E2 indicating anomalous electrophoretic migration as described for the E2 components of other pyruvate dehydrogenase complexes. Thus, the organization and subunit Mr values are similar with the mammalian complexes and virtually identical with the complexes of gram-positive bacteria but differ greatly from the pyruvate dehydrogenase complexes of gram-negative bacteria. 3. The complex was resolved into its component enzymes by the following methods. E1 was obtained by treatment of the complex with elastase followed by gel chromatography on Sepharose CL-2B using a reverse ammonium sulfate gradient for elution. E2 was isolated by gel filtration of the complex in the presence of 2 M KBr, and E3 was obtained by hydroxyapatite chromatography in 8 M urea. The isolated enzymes reassociated spontaneously to give pyruvate dehydrogenase overall activity.
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PMID:Pyruvate dehydrogenase complex from baker's yeast. 2. Molecular structure, dissociation, and implications for the origin of mitochondria. 703 Jul 41

A novel dihydrolipoyl dehydrogenase-binding protein (E3BP) which lacks an amino-terminal lipoyl domain, p45, has been identified in the pyruvate dehydrogenase complex (PDC) of the adult parasitic nematode, Ascaris suum. Sequence at the amino terminus of p45 exhibited significant similarity with internal E3-binding domains of dihydrolipoyl transacetylase (E2) and E3BP. Dissociation and resolution of a pyruvate dehydrogenase-depleted adult A. suum PDC in guanidine hydrochloride resulted in two E3-depleted E2 core preparations which were either enriched or substantially depleted of p45. Following reconstitution, the p45-enriched E2 core exhibited enhanced E3 binding, whereas, the p45-depleted E2 core exhibited dramatically reduced E3 binding. Reconstitution of either the bovine kidney or A. suum PDCs with the A. suum E3 suggested that the ascarid E3 was more sensitive to NADH inhibition when bound to the bovine kidney core. The expression of p45 was developmentally regulated and p45 was most abundant in anaerobic muscle. In contrast, E3s isolated from anaerobic muscle or aerobic second-stage larvae were identical. These results suggest that during the transition to anaerobic metabolism, E3 remains unchanged, but it appears that a novel E3BP, p45, is expressed which may help to maintain the activity of the PDC in the face of the elevated intramitochondrial NADH/NAD+ ratios associated with anaerobiosis.
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PMID:Identification of a novel dihydrolipoyl dehydrogenase-binding protein in the pyruvate dehydrogenase complex of the anaerobic parasitic nematode, Ascaris suum. 862 1

Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli. The E2 component was purified as a large soluble aggregate (molecular mass > 1 x 10(6) Da) with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa. The recombinant E2 component in vitro was capable of binding either 60 E3(alpha2) dimers or 60 heterotetramers (alpha2beta2) of the pyruvate decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain. This has important consequences for the stoichiometry of the assembled complex in vivo. The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post-translationally modified in vitro using a recombinant lipoate protein ligase from E. coli. The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild-type enzyme. Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95% and 85%, respectively. However, only 26% of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1alpha and E1beta. This represents the first complete post-translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.
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PMID:Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro. 987 16


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