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

---

Query: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Results are presented which demonstrate that the 2-electron-reduced lipoamide dehydrogenase (EC 1.6.4.3) from Escherichia coli is a mixture of species. In catalysis, this enzyme cycles between the oxidized and the 2-electron-reduced forms. Three spectrally distinct species are indicated in the pH range 5.8 to 8.0 from measurements of the fluorescence and visible spectra during dithionite titration. These have the following properties. 1) A fluorescent form where the FAD is oxidized and the active center disulfide is reduced. This species is unable to charge transfer and predominates at low pH. 2) A form in which there is a facile charge transfer between thiolate and FAD (epsilon530 - 3300 M-1 cm-1). This species, which predominates at high pH, is very similar to the 2-electron-reduced pig heart enzyme at high pH. 3) A form where the flavin is reduced and the disulfide is oxidized. The spectra of these three species have been determined. Anaerobic reduction of the enzyme with stoichiometric dihydrolipoamide leads to the formation of the charge transfer species in less than 1 s. Subsequently, in a process requiring about 12 s, the charge transfer complex relaxes to a mixture of species observed in dithionite titrations. The pH dependence of the oxidation-reduction potential, the fluorescence, the charge transfer absorbance (530 nm), and the 455 nm absorbance indicates the presence of a base which is able to stabilize the thiolate anion generated upon reduction of the active center disulfide. The pH dependence of the oxidation-reduction potential indicates that the reduction of the enzyme by dihydrolipoamide involves 2 protons as well as 2 electrons. These potentials are somewhat more positive than those determined for the pig heart enzyme and thus explain the ready further reduction of the E. coli enzyme to the 4-electron-reduced enzyme. The pH-independent formation constant (Kf) for the disproportionation of 2-electron-reduced enzyme (2EH2 in equilibrium E + EH4) is about 55 as calculated from dithionite titrations. Therefore at equilibrium there is about 80% 2-electron-reduced enzyme, 1-% oxidized enzyme, and 10% 4-electron-reduced enzyme. The spectrum of fully formed 2-electron-reduced enzyme has been calculated at several pH values from these data. The results confirm the previous conclusion that lipoamide dehydrogenase from E. coli is qualitatively similar to the pig heart enzyme, differing only in certain quantitative features such as the distribution between the various forms at the 2-electron-reduced level.
...
PMID:Evidence for multiple electronic forms of two-electron-reduced lipoamide dehydrogenase from Escherichia coli. 3 77

Labelling studies with N-ETHYLMALEIMIDE SHOW THAT EITHER IN THE PRESENCE OF Mg2+, thiamine pyrophosphate (TPP) and pyruvate or in the presence of NADH the overall activity of the pyruvate dehydrogenase complex from Azotobacter vinelandii is inhibited without much inhibition of the partial reactions. The complex undergoes a conformational change upon incubation with NADH. The inhibition by bromopyruvate is less specific. Specific incorporation of a fluorescent maleimide derivative was observed on the two transacetylase isoenzymes. Binding studies with a similar spin label analogue show that 3 molecules/FAD are incorporated by incubation of pyruvate, Mg2+ and TPP, whereas 2 molecules/FAD are incorporated via incubation with NADH. The spin label spectra support the idea that in the complex the active centres of the component enzymes are connected by rapid rotation of the lipoyl moiety. Three acetyl groups are incorporated in the complex by incubation with [2-14C]pyruvate. Time-dependent incorporation supports the view that the two transacetylase isoenzymes react in non-identical ways with the pyruvate dehydrogenase components of the complex. The results show that the complex contains 2 low-molecular-weight transacetylase molecules and 4 molecules of the high-molecular-weight isoenzyme. Mn2+-binding studies show that the complex binds 10 ions, with different affinities. 2 Mn2+ ions are bound with a 20-fold higher affinity than the remaining 8 Mn2+ ions. The latter 8 ions bind with equal affinities and are thought to reflect binding to the pyruvate dehydrogenase components of the complex. It is concluded that the complex contains 8 pyruvate dehydrogenase molecules, 4 high-molecular-weight transacetylase molecules, 2 low-molecular-weight transacetylase molecules and 1 dimeric (2-FAD-containing) symmetric molecule of lipoamide dehydrogenase. Evidence comes from pyruvate-dependent inactivation and labelling studies that the pyruvate dehydrogenase components contain either an - SH group or an S-S bridge which participates in the hydroxyethyl transfer to the transacetylase components.
...
PMID:The pyruvate-dehydrogenase complex from Azotobacter vinelandii. 3. Stoichiometry and function of the individual components. 17 36

Asparagusate dehydrogenases I and II and lipoyl dehydrogenase have been obtained in homogeneous state from asparagus mitochondria. They are flavin enzymes with 1 mol of FAD/mol of protein. Asparagusate dehydrogenases I and II and lipoyl dehydrogenase have s20,w of 6.22 S, 6.39 S, and 5.91 S, respectively, and molecular weights of 111,000, 110,000, and 95,000 (sedimentation equilibrium) or 112,000, 112,000, and 92,000 (gel filtration). They are slightly acidic proteins with isoelectric points of 6.75, 5.75, and 6.80. Both asparagusate dehydrogenases catalyzed the reaction Asg(SH)2 + NAD+ equilibrium AsgS2 + NADH + H+ and exhibit lipoyl dehydrogenase and diaphorase activities. Lipoyl dehydrogenase is specific for lipoate and has no asparagusate dehydrogenase activity. NADP cannot replace NAD in any case. Optimum pH for substrate reduction of the three enzymes are near 5.9. Asparagusate dehydrogenases I and II have Km values of 21.5 mM and 20.0 mM for asparagusate and 3.0 mM and 3.3 mM for lipoate, respectively. Lipoyl dehydrogenase activity of asparagusate dehydrogenases is enhanced by NAD and surfactants such as lecithin and Tween 80, but asparagusate dehydrogenase activity is not enhanced. Asparagusate dehydrogenases are strongly inhibited by mercuric ion, p-chloromercuribenzoic acid, and N-ethylmaleimide. Amino acid composition of the three enzymes is presented and discussed.
...
PMID:Asparagusate dehydrogenases and lipoyl dehydrogenase from asparagus mitochondria. Physical, chemical, and enzymatic properties. 18 3

A derivative of the flavoprotein pig heart lipoamide dehydrogenase has been described recently (Thorpe, C., and Williams, C.H. (1976) J. Biol. Chem. 251, 3553-3557), in which 1 of the 2 cysteine residues generated on reduction of the intrachain active center disulfide bridge is selectively alkylated with iodoacetamide. This monolabeled enzyme exhibits a spectrum of oxidized bound flavin. The addition of 1 mM NAD+ to this derivative at pH 8.3 causes a decrease in absorbance of approximately 50% at 448 nm, with a concomitant increase at 380 nm. These spectral changes are complete within 3 ms and are reversible. NAD+ titrations generate isosbestic points at 408, 374, and 327 nm; allowing values for the apparent dissociation constant for NAD+ and the extent of bleaching at infinite ligand to be obtained from double reciprocal plots. Between pH 6.1 and 8.8, the apparent KD decreases from 320 to 35 muM, whereas the extrapolated delta epsilon 448 values remain approximately constant at 1/2 epsilon 448. Direct measurement of NAD+ binding by gel filtration at pH 8.8 indicates that the spectral changes are associated with a stoichiometry of 1.2 mol of NAD+ bound/2 mol of FAD. The modified protein is a dimer containing 1 FAD and 1 alkylated cysteine residue/subunit; the native enzyme is also dimeric. The visible spectrum of the species absorbing at 380 nm, approximated by correction for the residual oxidized FAD, shows a single maximum at 384 nm, epsilon 384 = 8.7 mM-1cm-1. Comparison of this spectrum with that of model compounds of known structure suggests that it may represent a reversible covalent flavin adduct induced on binding NAD+.
...
PMID:Spectral evidence for a flavin adduct in a monoalkylated derivative of pig heart lipoamide dehydrogenase. 18 94

Fluorescence energy transfer has been employed to estimate the minimum distance between each of the active sites of the 4 component enzymes of the pyruvate dehydrogenase multienzyme complex from Azotobacter vinelandii. No energy transfer was seen between thiochrome diphosphate, bound to the pyruvate decarboxylase active site, and the FAD of the lipoamide dehydrogenase active site. Likewise, several fluorescent sulfhydryl labels, which were specifically bound to the lipoyl moiety of lipoyl transacetylase, showed no energy transfer to either the flavin or thiochrome diphosphate. These observations suggest that all the active centers of the complex are quite far apart (greater than or equal to 40 nm), at least during some stages of catalysis. These results do not preclude the possibility that the distances change during catalysis. Several of the fluorescent probes used possessed multiple fluorescent lifetimes, as shown by determination of lifetime averages by both phase and modulation measurements on a phase fluorimeter. These lifetimes are shown to result from multiple factors, not necessarily related to multiple protein conformations.
...
PMID:Fluorescence energy-transfer studies on the pyruvate dehydrogenase complex isolated from Azotobacter vinelandii. 34 64

The dynamic structures of two major forms (LD(I) and LD(II) of pig heart lipoamide dehydrogenase, resolved by TEAE-cellulose column chromatography, were studied by fluorescence depolarization. FAD and ANM were used as an intrinsic and an extrinsic fluorescent probe, respectively. In the experiments with bound FAD of lipoamide dehydrogenase, no thermal dependence of the fluorescence depolarization of either enzyme was observed and the values of polarization were close to the theoretical maximum value of 0.5. Both enzymes contained two reactive thiol groups which differed in their reactivities toward ANM. When the enzymes were labeled with one mol of ANM per mol of enzyme, the rotational relaxation times of LD(I) and LD(II) were found to be 18 ns and 196 ns, respectively. These findings indicate that the sement of LD(I) labeled with ANM fluctuates in the order of nanoseconds, whereas this segment of LD(II) is fixed rigidly. On the other hand, when the enzymes were labeled with two mol of ANM per mol of enzyme, both enzymes showed the composite result of fluorescence depolarization due to the motilities of the segment of enzyme and the whole enzyme molecule. These findings indicate that both LD(I) and LD(II) have the non-equivalent motilities of segments containing one reactive thiol group between the two monomers. In other words, the segment containing the ANM binding site of the one monomer is flexible and this segment of the other monomer is fixed rigidly in both enzymes.
...
PMID:Fluorescence studies on lipoamide dehydrogenases of pig heart. I. Conformational dynamics of enzyme. 50 May 85

A method is described for preparation of apolipoamide dehydrogenase which gives quantitative removal of FAD. Active holoenzyme can be reconstituted by incubation with FAD. Reconstitution of apoenzyme with 8-Cl-FAD results in the fixation of most of the flavin to the protein in a covalently bound form. The portion noncovalently bound was shown to be unmodified 8-Cl-FAD. The covalently bound flavin has an absorption spectrum quite different from that of 8-Cl-FAD. It has a single band in the visible with a maximum at 459 nm (extinction coefficient of 22 mM-1 cm-1) and a shoulder at 480 nm. Model reactions between 8-Cl-Flavin (riboflavin or FAD) and organic thiols (thiophenol, beta-mercaptoethanol, or N-acetylcysteine) give products with spectra which are similar to that of FAD covalently bound to lipoamide dehydrogenase. The products of the model reactions have a single visible band with a maximum at 480 nm (extinction coefficient of 23.6 mM-1 cm-1 to 28.4 mM-1 cm-1) and a shoulder at 460 nm. The products of the model reaction and the covalently bound FAD of lipoamide dehydrogenase appear to be the result of a nucleophilic attack on the carbon at position 8 of the flavin ring by a thiolate anion, displacing the chloride. Thus, the product of the model reaction is 8-(RS)-flavin, and the product of the reaction between 8-Cl-FAD and protein probably has a cysteinyl residue covalently attacked at position 8 of FAD. Reconstitution of apoliopoamide dehydrogenase with 8-Cl-FAD gives two enzyme products which are fractionated by ammonium sulfate. Enzyme fractionating between 20% and 45% ammonium sulfate is monomeric and contains covanently bound FAD. Enzyme fractionating between 55% and 75% ammonium sulfate is dimeric and contains both covalently bound FAD and noncovalently bound 8-Cl-FAD. Both protein fractions contain one FAD per protein subunit and both are active with physiological substrates with Km values for NAD and dihydrolipoamide similar to those of native lipoamide dehydrogenase. The maximum turnover rates differ dramatically. Enzyme fractionating between 55% and 75% ammonium sulfate has a Vmax which is 61 times slower than native enzyme. Enzyme fractionating between 20% and 45% ammonium sulfate has a Vmax which is 7400 times slower than native enzyme. These slower rates are partially explainable by the oxidation-reduction potentials of the modified enzymes. Both covalently bound FAD and noncovalently bound FAD appear to reside in the native flavin binding site of the enzyme. However, once dimerization of the protien has taken place, the noncovalently bound 8-Cl-FAD cannot be induced to form a covalent bond with the protein except under protein denaturing conditions. The implications of these findings are discussed.
...
PMID:Production of a covalent flavin linkage in lipoamide dehydrogenase. Reaction with 8-Cl-FAD. 68 58

The interaction of the pyruvate dehydrogenase multienzyme complex from Escherichia coli with 8-anilino-1-naphthalenesulfonate (ANS), pyruvate, and acetyl-CoA has been investigated using equilibrium binding, steady-state fluorescence, and fluorescence lifetime measurements. The fluorescnece of ANS is greatly enhanced when bound to the enzyme complex and to the pyruvate dehydrogenase component of the complex. Approximately 22 molecules of ANS are bound to a molecule of the complex with a binding constant of 3.69 muM in 0.03 M potassium potassium phosphate (pH 7.0). Direct and competitive binding measurements indicate that about 42 pyruvate binding sites are present per mole of enzyme complex which has been stripped of thiamine diphosphate; the number of binding sites is reduced to 28,5 in the presence of a saturating concentration of thiochrome diphosphate, a thiamine diphosphate analogue. The dissociation constant for pyruvate to the enzyme complex in the presence of thiochrome diphosphate is 308 muM in 0.02 M potassium phosphate (pH 7.0). Pyruvate, thiochrome diphosphate, and acetyl-CoA all displace ANS from the enzyme complex. In the cases of pyruvate and thiochrome diphosphate, the concentration dependence of the displacements suggests the displacement is allosteric, while in the case of acetyl-CoA direct competition appears to be involved. GTP decreased the effect of acetyl-CoA to the enzyme complex indicate that 24-26 bound acetyl-CoA molecules per complex can be readily displaced by ANS, and the binding of acetyl-CoA to these sites displays positive cooperativity. Fluorescence energy transfer measurements between bound ANS on the pyruvate dehydrogenase enzyme and FAD on the dihydrolipoyl dehydrogenase enzyme indicate, assuming the emission and absorption dipoles are randomly oriented, that these two probes must be at least 58 A apart in the intact complex.
...
PMID:Fluorescence energy transfer measurements between ligand binding sites of the pyruvate dehydrogenase multienzyme complex. 76 64

Fluorescence-lifetime measurements of FAD bound to lipoamide dehydrogenase from Azotobacter vinelandii and Escherichia coli were performed. It is shown from these results that the two FAD groups in the isolated dimeric enzyme, as well as in the enzyme in the intact complex of E. coli, are in non-equivalent surroundings. This contrasts with the near equivalence of the FAD groups of both the enzyme and complex isolated from A. vinelandii. Reduction of the complex with Mg2+, thiamine pyrophosphate and pyruvate or with NADH enables the attachment of a maleimide analogue specifically to the lipoyl moieties of the transacetylase(s). Spin label [N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide] introduced in such a way proves the existence of at least two different micro-environments around the lipoyl moieties in complex isolated from A. vinelandii. Electron paramagnetic resonance spectra of the specifically spin-labelled complexes from E. coli and A. vinelandii, when dissolved in tricine [N-tris(hydroxymethyl)-methylglycine] buffer, show interactions of at least two electron spins with each other, which indicate that the lipoyl moieties are rather close together. Fluorescent label [N-(1-anilinonaphthyl-4)maleimide] is specifically attached to the lipoyl moiety of the high-Mr transacetylase of the freshly isolated complex from A. vinelandii. From the large differences in the apparent lifetimes tau p and tau m, as detected by phase fluorimetry, it is shown that this fluorscent label is distributed in different micro-environments. The differences observed in energy transfer between fluorescent label, attached to the lipoyl moiety of the high-Mr transacetylase, indicate different conformations of the complex from A. vinelandii. Upon introduction of the label after reduction with NADH a much larger energy transfer, thus a shorter distance, is observed between the label and FAD than when reduction is performed with Mg2+, thiamine pyrophosphate and pyruvate. A similar conformation dependence upon reduction is found for the pyruvate dehydrogenase complex from E. coli. It is thus proposed that the transacetylase of E. coli and the high-Mr transacetylase of A. vinelandii are both non-symmetrically distributed within the complex.
...
PMID:Symmetry and asymmetry of the pyruvate dehydrogenase complexes from Azotobacter vinelandii and Escherichia coli as reflected by fluorescence and spin-label studies. 79 71

Microorganisms formed readily ethylenethiourea (ETU) from 5,6-dihydro-3H-imidazo[2,1-c]-1,2,4-dithiazole-3-thione (DIDT), a spontaneous decomposition product of ethylenebisdithiocarbamates. This conversion also takes place after addition of reducing compounds like cysteine, glutathione or ascorbic acid. It consists of two steps: reduction of the S-S bond of DIDT with subsequent release of CS2 to form ETU. DIDT was reduced by NADH in the presence of enzyme extracts from Pseudomonas fluorescens or Asperigillus niger, or by commercial glutathione reductase or lipoamide dehydrogenase. ETU was formed as a result of this enzymatic reduction. The flavin compounds FMN and FAD were also able to promote the reduction of DIDT by NADH.
...
PMID:Formation of ethylenethiourea from 5,6-dihydro-3H-imidazo[2,1-c]-1,2,4-dithiazole-3-thione by microorganisms and reducing agents. 81 82


1 2 3 4 5 6 7 8 9 10 Next >>

---