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: EC:1.8.1.4 (diaphorase)
2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced with other residues. His450, the active-site base, was replaced with Ser, Tyr or Phe. Pro451, from X-ray analysis found to be in cis conformation positioning the backbone carbonyl of His450 close to N3 of the flavin, was changed to Ala. Glu455, from X-ray analysis expected to be involved in modulating the pKa of the base (His450), was replaced with Asp and Gln. The general conclusion is that mutation of the His-Glu diad impairs intramolecular electron transfer between the disulfide/dithiol and the FADH-/FAD. The wild-type enzyme functions according to a ping-pong mechanism in the physiological reaction in which the formation of NADH is rate-limiting. Above pH 8.0 the enzyme is strongly inhibited by the product NADH. The pH dependence of the steady-state kinetics using the NAD+ analog 3-acetylpyridine adenine dinucleotide (AcPyAde+) reveals a pKa of 8.1 in the pKm AcPyAde+ plot indicating that this pKa is related to the deprotonation of His450 [Benen, J., Berkel van, W., Zak, Z., Visser, T., Veeger, C. & Kok de, A. (1991) Eur. J. Biochem. 202, 863-872] and to the inhibition by NADH. The mutations considerably affect turnover. Enzymes with the mutations Pro451----Ala, His450----Phe and His450----Tyr appear to be almost inactive in both directions. Enzyme His450----Ser is minimally active, V at the pH optimum being 0.5% of wild-type activity in the physiological reaction. Rapid reaction kinetics show that for the His450-mutated enzymes the reductive half reaction using reduced 6,8-thioctic acid amide [Lip(SH)2] is rate-limiting and extremely slow when compared using reduced 6,8-thioctic acid amide [Lip(SH)2] is rate-limiting and extremely slow when compared to the wild-type enzyme. For enzyme Pro451----Ala it is concluded that the loss of activity is due to over-reduction by Lip(SH)2 and NADH. The Glu455-mutated enzymes are catalytically competent but show strong inhibition by the product NADH (enzyme Glu455----Asp more than Glu455----Gln). The inhibition can largely be overcome by using AcPyAde+ instead of NAD+ in the physiological reaction. The rapid reaction kinetics obtained for enzymes Glu455----Asp and Glu455----Gln deviate from the wild-type enzyme. It is concluded that this difference is due to cooperativity between the active sites in this dimeric enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Lipoamide dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad. Kinetics of wild-type and mutated enzymes. 163 4

The 10 C-terminal residues are not visible in the crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii, but can be observed in the crystal structures of the lipoamide dehydrogenases from Pseudomonas putida and Pseudomonas fluorescens. In these structures, the C-terminus folds back towards the active site and is involved in interactions with the other subunit. The function of the C-terminus of lipoamide dehydrogenase from A. vinelandii was studied by deletion of 5, 9 and 14 residues, respectively. Deletion of the last 5 residues does not influence the catalytic properties and conformational stability (thermoinactivation and unfolding by guanidinium hydrochloride). Removal of 9 residues results in an enzyme (enzyme delta 9) showing decreased conformational stability and high sensitivity toward inhibition by NADH. These features are even more pronounced after deletion of 14 residues (enzyme delta 14). In addition Tyr16, conserved in all lipoamide dehydrogenases sequenced thus far, and shown from the other structures to be likely to be involved in subunit interaction, was replaced by Phe and Ser. Mutation of Tyr16 also results in a strongly increased sensitivity toward inhibition by NADH. The conformational stability of both Tyr16-mutated enzymes is comparable to enzyme delta 9. The results strongly indicate that a hydrogen bridge between tyrosine of one subunit (Tyr16 in the A. vinelandii sequence) and histidine of the other subunit (His470 in the A. vinelandii sequence), exists in the A. vinelandii enzyme. In the delta 9 and delta 14 enzymes this interaction is abolished. It is concluded that this interaction mediates the redox properties of the FAD via the conformation of the C-terminus containing residues 450-470.
...
PMID:Lipoamide dehydrogenase from Azotobacter vinelandii. The role of the C-terminus in catalysis and dimer stabilization. 163 5

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced by other residues. His450, the active-site base, was changed into Ser, Tyr and Phe. Pro451, in cis conformation, was changed into Ala. Glu455 was replaced with Asp and Gln. Absorption, fluorescence and CD spectroscopy of the mutated enzymes in their oxidized state (Eox) showed only minor changes with respect to the wild-type enzyme, whereas considerable changes were observed in the spectra of the two-electron-reduced (EH2) species of the enzymes upon reduction by the substrate dihydrolipoamide. Differences in extent of reduction of the flavin by NADH indicate that the redox potential of the flavin is altered by the mutations. Enzyme Pro451----Ala [corrected] showed the greatest deviation from wild type. The enzyme is very easily over-reduced to the four-electron reduced state (EH4) by dihydrolipoamide. This is probably due to a change in the backbone conformation caused by the cis-trans conversion. From studies on the pH dependence of the thiolate charge-transfer absorption and the relative fluorescence of EH2 of the enzymes, it is concluded that mutation of His450 results in a relatively simple and easily interpreted distribution of electronic species at the EH2 level. For all three His450-mutated enzymes an apparent pKa1 near 5.5 is calculated that is assigned to the interchange thiol. A second apparent pKa2 is calculated of 6.9, 7.5 and 7.1 for the His450----Phe, -Ser and -Tyr enzymes, respectively, and signifies the deprotonation of the tautomeric equilibrium between the interchange and charge-transfer thiols. The difference in apparent pKa2 values between the His450-mutated enzymes is explained by changes in micropolarity. At the EH2 level the wild-type enzyme consists of multiple electronic forms as reported for the Escherichia coli enzyme [Wilkinson, K. D. and Williams C. H. Jr (1979) J. Biol. Chem. 254, 852-862]. Based on the results obtained with the His450-mutated enzymes, it is concluded that the lowest pKa is associated with the interchange thiol. A model for the equilibrium species of the wild-type enzyme at the EH2 level is presented which takes three pKa values into account. The results of the pH dependence of the electronic species at the EH2 level of Glu455-mutated enzymes essentially follow the model proposed for the wild-type enzyme. However mutation of Glu455 shifts the tautomeric equilibrium of EH2 in favor of the charge-transfer species.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Lipoamide dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad. Spectral properties of wild type and mutated enzymes. 168 37

A simple, rapid, accurate, and precise colorimetric assay for the determination of L-phenylalanine in plasma samples using L-phenylalanine dehydrogenase [L-phenylalanine:NAD+-oxidoreductase (deaminating)] from Rhodococcus sp. M 4 is described. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine. However, the equilibrium of reaction favors L-phenylalanine formation. By stoichiometric coupling of this reaction with diaphorase/iodonitro tetrazolium chloride (INT) the formed NADH converts INT to a formazan whereby the reaction is displaced in favor of phenylpyruvate. Using a kinetic approach the increase in absorbance at 492 nm shows linearity over more than 30 min. Deproteinized standard solutions of L-phenylalanine in the range from 30 to 1200 mumol/liter show a linearity between the dAformazan/30 min and the substrate concentration. In phenylketonuria (PKU) plasma samples no interferences caused by L-tyrosine or phenylpyruvic acid are seen. Applicability is demonstrated by comparative determination of plasma L-phenylalanine of treated PKU patients by the colorimetric method and automated amino acid analysis.
...
PMID:Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase. 281 48

The carboxyl-terminal region of plant ferredoxin-NADP+ reductases is formed by an invariant alpha-helix/loop/beta-strand, culminating in a conserved tyrosine that displays extensive interaction with the prosthetic group FAD. We have investigated the potential role of the terminal region in reductase function, by introducing mutations and deletions on pea ferredoxin-NADP+ reductase overexpressed in Escherichia coli. Replacement of the terminal tyrosine by tryptophan, phenylalanine, serine, and glycine resulted in a 2.2-, 2.0-, 22-, and 302-fold reduction, respectively, in kcat for the diaphorase reaction, whereas elimination of the tyrosine caused a 846-fold decrease in kcat. Km values were largely unaffected by the substitutions. Similar results were obtained when the mutants were assayed for cytochrome c reduction, indicating that aromaticity is the most important factor to the function of the tyrosine in catalysis. The presence of the phenol ring at the carboxyl-terminal position of wild-type reductase is important, but not an absolute requirement for enzyme function or FAD assembly. Deletion of the alpha-helix/beta-strand region prevented reductase proper folding in the bacterial host, while shortening of the terminal region by splicing 3 amino acids at the beginning of the alpha-helix produced a moderately soluble reductase, devoid of FAD and enzymatic activity.
...
PMID:Probing the role of the carboxyl-terminal region of ferredoxin-NADP+ reductase by site-directed mutagenesis and deletion analysis. 836 77

Systematic replacement of a set of amino acids in the beta alpha beta-fold of the NAD-binding domain of Escherichia coli dihydrolipoamide dehydrogenase has been used to convert its coenzyme specificity from NAD to NADP. After comparison with the homologous enzyme glutathione reductase, Glu 203 was replaced with a valine residue, thereby eliminating the potential to form hydrogen bonds with the 2'- and 3'-OH groups of the adenine ribose in NAD. Similarly, Met 204, Pro 210, Phe 205, and Asp 206 were replaced by an arginine, an arginine, a lysine, and a histidine residue, respectively, to provide a nest of positive charge to accommodate the 2'-phosphate group of the incoming NADP. In addition, Gly 185 and Gly 189 in the beta alpha beta motif were replaced with alanine residues to facilitate the positioning of the newly introduced Val 203 by allowing a flip of the peptide bond between residues Gly 180 and Gly 181. Wild-type dihydrolipoamide dehydrogenase is inactive with NADP, but the mutant enzyme displayed high levels of activity with this coenzyme, the values of Km, kcat, and kcat/Km comparing favorably with those found for the wild-type enzyme operating with NAD. The mutant enzyme was also capable of assembly in vitro to form an active pyruvate dehydrogenase multienzyme complex, the coenzyme specificity of which reflected that of its dihydrolipoamide dehydrogenase component. These experiments should make it possible now to study the effects in vivo of requiring a crucial catabolic enzyme to function with the wrong coenzyme, an important extension of protein engineering into the living cell.
...
PMID:Creation of an NADP-dependent pyruvate dehydrogenase multienzyme complex by protein engineering. 845 41

The sensitivity of lipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase E3) from Azotobacter vinelandii to inhibition by NADH requires measurement of the activity in the initial phase of the reaction. Stopped-flow turnover experiments show that kcat is 830 s-1 compared with 420 s-1 found in standard steady-state experiments. Mutations at the si-side of the flavin prosthetic group that cause severe inhibition by NADH were studied. Tyr16 was replaced by phenylalanine and serine, which causes the loss of two intersubunit H-bonds. [F16]E3 shows only 5.7% of wild-type activity in the standard assay procedure, but analyzed by stopped-flow the activity is 70% of the wild-type enzyme. The NADH-->Cl2Ind (dichloroindophenol) activity was normal or slightly increased. The inhibition by NADH is competitive with respect to NAD+, Ki = 50 microM. Spectral analysis show that electrons readily pass over from the disulfide to the FAD, indicating an increase in the redox potential of the flavin. It is concluded that subunit interaction plays an important role in the protection of the enzyme against over-reduction by decreasing the redox potential of the flavin. The interaction of wild-type or mutant enzymes with the core component of the pyruvate (E2p) or oxoglutarate (E2o) dehydrogenase multienzyme complex relieves the inhibition to a large extent. In the mutant enzymes, the mechanism of inhibition changes from competitive to the mixed-type inhibition observed for the wild-type enzyme. The stabilizing effect of E2 on [F16]E3 was used as an assay to analyze the stoichiometry of interaction of E3 with E2p as well as E2o. 1 mol E2p monomer was sufficient to saturate 1 mol E3 dimer with a Kd of about 1 nM. Similarly, 1 mol E2o saturated the E3 dimer with a Kd of 30 nM. From these experiments it is concluded that the E3-binding domain of E2 interacts with the subunit interface of E3 near the dyad axis, thus preventing sterically the interaction with a second molecule of the binding domain. This mode of interaction, which causes asymmetry in the complex, explains the stabilization against over-reduction by tightening the subunit interaction. Subgene cloning of the E2p component of the pyruvate dehydrogenase complex is described in order to obtain a complex between the lipoamide dehydrogenase component (E3) and the binding domain of E2p. A unique restriction site in the DNA encoding the flexible linker between the third lipoyl domain and the binding domain combined with timed digestion with exonuclease Bal31 was used to create a set of deletion mutants in the N-terminal region of the binding-catalytic didomain, fused to six N-terminal amino acids from beta-galactosidase. The expressed proteins, selected for E2p activity, were analyzed for binding of E3 and E1p. The shortest fusion protein containing a functional binding domain was expressed and purified. [F16]E3 was combined with this fusion protein in a stoichiometric ratio and the resulting complex was subjected to limited proteolysis to remove the catalytic domain. The resulting [F16]E3-binding domain preparation was purified to homogeneity.
...
PMID:The interaction between lipoamide dehydrogenase and the peripheral-component-binding domain from the Azotobacter vinelandii pyruvate dehydrogenase complex. 857 46

The occurrence of NADH --> NAD transhydrogenation and lipoamide dehydrogenase activities was demonstrated for cysticercoids of the intestinal cestode, Hymenolepis diminuta. In addition, both activities were catalyzed by the mitochondria of 6-, 10-, and 14-day H. diminuta and by the mitochondria from immature, mature, and pregravid/gravid regions of the adult cestode. A developmentally related increase in NADH --> NAD activity was suggested and the levels of both activities in the immature region of the helminth were consistent with it being a region of high metabolic activity. Adult H. diminuta mitochondrial lipoamide dehydrogenase was purified to homogeneity. The native enzyme was a homodimer with a monomeric and dimeric molecular mass of 47 and 93 kDa, respectively. Spectral analyses revealed that the enzyme contained flavin. More importantly, the purified enzyme catalyzed appreciable NADH --> NAD transhydrogenation activity, a premier finding for the phylum Platyhelminthes. The ratio of NADH --> NAD transhydrogenation to lipoamide reduction was 1:5. Both activities were inhibited by Cu2+ and Cd2+ with the NADH --> NAD activity being more resistant to inhibition. Interestingly, aside from NADH diaphorase activity, the cestode enzyme displayed NADH-ferricyanide reductase and, to a lesser degree, NADPH --> NAD transhydrogenation activities. The partial amino acid sequence of H. diminuta lipoamide dehydrogenase indicated that this enzyme was most similar to the corresponding enzymes of other parasitic helminths. Moreover, the phenylalanine for leucine substitution found in the redox-active disulfide site of the lipoamide dehydrogenases of some anaerobic systems was noted for the H. diminuta enzyme.
...
PMID:Hymenolepis diminuta: mitochondrial NADH --> NAD transhydrogenation and the lipoamide dehydrogenase system. 903 Jun 66

The retinopetal neurons of Crocodylus niloticus were visualized by retrograde transport of rhodamine beta-isothiocyanate or Fast Blue administered by intraocular injection. Approximately 6,000 in number, these neurons are distributed in seven regions extending from the mesencephalic tegmentum to the rostral rhombencephalon, approximately 70% being located contralaterally to the injected eye. None of the centrifugal neurons projects to both retinae. The retinopetal neurons are located in rostrocaudal sequence in seven regions: the formatio reticularis lateralis mesencephali, the substantia nigra, the griseum centralis tectalis, the nucleus subcoeruleus dorsalis, the nucleus isthmi parvocellularis, the locus coeruleus, and the commissura nervi trochlearis. The greatest number of cells (approximately 93%) is found in the nucleus subcoeruleus dorsalis. The majority are multipolar or bipolar in shape and resemble the ectopic centrifugal visual neurons of birds, although a small number of monopolar neurons resembling those of the avian isthmo-optic nucleus may also be observed. A few retinopetal neurons in the griseum centralis tectalis were tyrosine hydroxylase (TH) immunoreactive. Moreover, in the nuclei subcoeruleus dorsalis and isthmi parvocellularis, both ipsilaterally and contralaterally, approximately one retinopetal neuron in three (35%) was immunoreactive to nitric oxide synthase (NOS), and a slightly higher proportion (38%) of retinopetal neurons were immunoreactive for choline acetyltransferase (ChAT). Some of them contained colocalized ChAT and NOS/reduced nicotinamide adenine dinucleotide phosphate-diaphorase. Fibers immunoreactive to TH, serotonin (5-HT), neuropeptide Y (NPY), or Phe-Met-Arg-Phe-amide (FMRF-amide) were frequently observed to make intimate contact with rhodamine-labeled retinopetal neurons. These findings are discussed in relation to previous results obtained in other reptilian species and in birds.
...
PMID:Centrifugal visual system of Crocodylus niloticus: a hodological, histochemical, and immunocytochemical study. 1464 91

We describe here a new microquantification method of l-phenylalanine concentration in an extract from a dried blood spot by using the diaphorase-resazurin system. To miniaturize the fluorometric enzymatic microplate assay for the diagnosis of phenylketonuria, an enzyme chip immobilized with His-tag fused phenylalanine dehydrogenase (PheDH) was developed. His-tag fused PheDH was immobilized on the surface of nickel-coated slide glass. A microarray sheet (8 x 30 well) was fabricated with poly(dimethylsiloxane) (PDMS) using the photolithographic technique. An enzyme reaction chamber in a double-layered structure was constructed with different types of microarray PDMS sheets on the surface of Ni-coated slide glass immobilized with His-tagged PheDH. To evaluate the affinity toward the Ni-chelating ligand, eight kinds of His-tagged PheDH variants were constructed and expressed. (His)(6)- and (His)(9)-PheDH variants at the N terminus showed high adsorption ratio to Ni-chelating ligand. The V(max) and k(cat) values of the (His)(6)-PheDH variant at the N terminus for l-phenylalanine were higher than those of the (His)(9)-PheDH variant, and the (His)(6)-PheDH variant was found to be most suitable for immobilization onto nickel-coated slide glass. Fluorescence formed by resazurin-coupled enzymatic reaction (in a 0.2-microl reaction mixture) on the enzyme chip exhibited good linearity and a correlation coefficient up to 12.8 mg/dl of the l-phenylalanine-containing sample extracted from a dried blood spot on filter paper.
...
PMID:Application of an enzyme chip to the microquantification of l-phenylalanine. 1704 6


1 2 Next >>

---