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)

Reduction of the active center disulfide bond in the flavoprotein pig heart lipoamide dehydrogenase generates two sulfur moieties which are chemically inequivalent in the 2-electron reduced form of the enzyme. Thus 1 cysteine residue is at least 13-fold more reactive than its partner toward iodoacetamide at pH 7.6. This selectivity was demonstrated by reaction of the 2-electron reduced enzyme with a low concentration of iodo[1-14C]acetamide under anaerobic conditions. The formation of a monolabeled derivative is accompanied by the reappearance of a spectrum of oxidized bound flavin, clearly different from that of the native enzyme. Alkylation of the remaining cysteine residues with iodo[12C]acetamide enabled the isolation of a tryptic version of the active center disulfide peptide. A single chymotryptic cleavage between the 2 alkylated cysteine residues generated a cationic and an anionic fragment containing 7% and 93% of the radioactivity of the purified tryptic peptide, respectively. The monolabeled derivative is catalytically inactive toward reduced or oxidized lipoamide, but is approximately 2-fold better as a transhydrogenase than the native protein using NADH and acetylpyridine adenine dinucleotide as substrates. Anaerobic titration with NADH leads to reduction of the flavin with concomitant formation of long wavelength absorption of low intensity. No intermediate reduced states were detected in this titration analogous to the red 2-electron form observed with the native enzyme. Similarly, intermediates during reduction of the enzyme by 1 eq of dithionite have not been detected.
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PMID:Differential reactivity of the two active site cysteine residues generated on reduction of pig heart lipoamide dehydrogenase. 0 57

The oxidation-reduction potential, E2, for the couple oxidized lipoamide dehydrogenase/2-electron reduced lipoamide dehydrogenase has been determined by measurement of equilibria of these enzyme species with lipoamide and dihydrolipoamide or with oxidized and reduced azine dyes. E2 is -0.280 V at pH 7, and deltaE2/deltapH is -0.06 V in the pH range 5.5 to 7.6. Values for E1, the oxidation-reduction potential for the couple 2-electron reduced enzyme/4-electron reduced enzyme, were obtained from measurements of the extent of dismutation of 2-electron reduced enzyme to form mixtures containing oxidized and 4-electron reduced enzyme. E1 is -0.346 V at pH 7, and deltaE1/deltapH is -0.06 V in the pH range 5.7 to 7.6. Spectra of oxidized enzyme and 4-electron reduced enzyme do not show variations with pH over this range, but the spectrum of the 2-electron reduced enzyme is pH-dependent, with the molar extinction at 530 nm changing from 3250 M-1 cm-1 at pH 8 to 2050 M-1 cm-1 at pH 5.2. The pH-dependent changes which are observed in the absorption properties of the 2-electron reduced enzyme are consistent with the disappearance of a charge transfer complex between an amino acid side chain and the oxidized flavin at the lower pH values, with the apparent pK of the side chain at pH 5. It has been suggested that the 530 nm absorbance of 2-electron reduced enzyme is due to a charge transfer complex between thiolate anion and oxidized flavin, and we propose that the thiolate anion is stabilized by interaction with a protonated base. The thermodynamic data predict that the amount of 4-electron reduced enzyme formed when the enzyme is reduced by excess NADH will be pH-dependent, with the greatest amounts seen at low pH values. These data support earlier evidence (Matthews, R.G., Wilkinson, K.D., Ballou, D,P., and Williams, C.H., Jr. (1976) in Flavins and Flavoproteins (Singer, T.P., ed) pp. 464-472; Elsevier Scientific Publishing Co., Amsterdam) that the role of NAD+ in the NADH-lipoamide reductase reaction catalyzed by lipoamide dehydrogenase is to prevent accumulation of inactive 4-electron reduced enzyme by simple reversal of the reduction of 2-electron reduced enzyme by NADH.
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PMID:Measurement of the oxidation-reduction potentials for two-electron and four-electron reduction of lipoamide dehydrogenase from pig heart. 0 67

A weak NADH oxidase activity of lipoamide dehydrogenase at neutral pH is increased as much as 15-fold by the addition of KI or (NH4)2SO4. The addition of NAD+ shifts the optimum pH for the KI-induced oxidase activity from 6.3 to 5.5 without changing the maximum activity. The optimum pH is similarly shifted to 5.6 when sulfhyldryl groups of the enzyme are oxidized in the presence of small amount of cupric ion. The NADH: lipoamide and NADH: p-benzoquinone reductase activities are strongly inhibited by KI but both are increased by the presence of (NH4)2SO4. The known intermediate having a charge-transfer band at 530 nm can be seen upon an addition of NADH to the enzyme in the presence of (NH4)2SO4 but not in the presence of KI. The enzyme flavin is reductase by a stoichiometric amount of NADH when KI is present.
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PMID:Salts- induced oxidase activity of lipoamide dehydrogenase from pig heart. 3 86

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

1. Pig heart lipoamide dehydrogenase (NADH: lipoamide oxidoreductase, EC 1.6.4.3) has been immobilised to Sepharose by thiol-disulphide interchange via a series of thiolated spacer molecules of increasing length. A number of properties of the immobilised enzyme have been investigated in order to ascertain the effects of proximity to the matrix backbone. 2. Proximity to the matrix backbone reduced the specific activity for lipoamide as substrate but enhanced by 3-8-fold the diaphorase activity with 2,6-dichloroindophenol. These observations are explained in part by an increase in the apparent Km for lipoamide when the enzyme is covalently attached to Sepharose via a short spacer molecule. 3. Both the thermal stability at 90 degrees C and the stability in 30% (v/v) dioxane are enhanced by up to 200% when the enzyme resides close to the matrix but approach those of the native enzyme as the length of the spacer molecule is increased. 4. These data have been correlated with measures of the accessibility of the enzyme as the nominal length of the spacer arm was increased. Thus, as the chain length increased, the rate of cleavage of the disulphide linkage between the enzyme and spacer increased and the enzyme became more susceptible to proteolysis by thermolysin. In contrast, increasing the chain length of the spacer made the enzyme less amenable to inhibition by a specific antibody. 5. These data are discussed in terms of the effect of the matrix on the conformation of the bound enzyme.
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PMID:Immobilised lipoamide dehydrogenase. 2. Properties of the enzyme immobilised to agarose through spacer molecules of various lengths. 56 Sep 66

Two unrelated patients with Friedreich ataxia were deficient in the activity of the enzyme lipoamide dehydrogenase (LAD). The enzymes from the patients' platelets differed significantly from controls in activity, in KM for lipoamide, and in KM for NADH. The data are consistent with a structural mutation of the gene coding for LAD.
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PMID:Kinetic evidence for a structural abnormality of lipoamide dehydrogenase in two patients with Friedreich ataxia. 56 87

The activity of lipoamide dehydorgenase (E.C.1.6.4.3) was measured in arterial homogenates from very young pigeons (5-8 weeks old) known to differ in their susceptibility to atherosclerosis. The activity of the arterial enzyme was significantly lower in the atherosclerosis-susceptible White Carneau pigeons than it was in the atherosclerosis-resistant Show Racer pigeons. Lipoamide dehydrogenase is a component of the pyruvate dehydrogenase and alpha-ketoglutarate multienzyme complexes. The first complex catalyzes the conversion of pyruvate to oxaloacetate via acetyl-CoA, and this reaction represents a crucial link between glycolysis and the Krebs cycle. The second complex is essential for the oxidative breakdown of carbohydrates, fats, and amino acids via the Krebs cycle. Reduced activity of these complexes, resulting from low activity of lipoamide dehydrogenase, favors reduction of pyruvate to lactate and a shift to glycolysis. This situation is in accord with other results obtained in avian and human arteries which appear to indicate a higher rate of glycolysis in atherosclerosis-susceptible and atherosclerotic arteries. It appears that the increased dependence of the White Carneau arteries on glycolysis, suggested by the reduced lipoamide dehydrogenase activity, facilitates the development of atherosclerosis in this pigeon strain.
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PMID:Inherited depression of arterial lipoamide dehydrogenase activity associated with susceptibility to atherosclerosis in pigeons. 112 73

The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2-fold axis generates the dimer which is observed in solution. The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 A2. The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region. The C-terminus has been proven to be important for activity by site-directed mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 A, and the precise role of the C-terminus still needs to be elucidated. In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 A for 440 well defined C alpha atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 A with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.
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PMID:The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution. 132 38

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


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