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:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In an attempt to establish the relative importance of diffusional and chemical control in the reactivity of the two of the two substrates, ubiquinol and cytochrome c, we have undertaken as extensive characterization of the steady-state kinetics of ubiquinol-cytochrome c reductase (EC 1.10.2.2) when present in open submitochondrial particles from bovine heart. The kinetic pattern follows a Ping Pong mechanism; contrary to the situation found with the isolated enzyme [Speck and Margoliash (1984) J. Biol. Chem. 259, 1064-1072, and confirmed in our laboratory], no substrate inhibition by oxidized cytochrome c was observed with the membrane-bound enzyme. Endogenous oxidized ubiquinone-10 is unable to exert product inhibition under the conditions employed. In the Ping Pong mechanism for this enzyme, the reaction scheme can be clearly divided into two parts, and the Kmin. (kcat./km) value for one substrate is independent of the rate constant for the second substrate. Both ubiquinol-1 and ubiquinol-2 can be used as electron donors reacting with the enzyme from within the lipid bilayer [Fato, Castelluccio, Palmer and Lenaz (1988) Biochim. Biophys. Acta 932, 216-222]; the kmin. values for ubiquinols, when calculated on the basis of their membranous concentrations, are significantly lower than the kmin. for cytochrome c. The temperature-dependence of the kinetic parameters was investigated by titrating each of the substrates under quasi-saturating concentrations of the second substrate. Arrhenius plots of Vmax. extrapolated from both cytochrome c and ubiquinol titrations were linear, when care was taken to verify the quasi-saturating concentrations of the fixed co-substrate. The Arrhenius plots for the kmin. values for both ubiquinol and cytochrome c were linear, but the activation energy was much higher for the former, particularly when calculated for ubiquinol dissolved in the lipid phase; the very low value of activation energy of the kmin. for cytochrome c is strong support for diffusion control being present in the reaction of cytochrome c with the membranous enzyme. In contrast to the soluble enzyme, ubiquinone titrations of submitochondrial particles at low cytochrome c concentrations deviated from hyperbolic behaviour. Changing the medium viscosity with either poly(ethylene glycol) or sucrose had a strong effect on the cytochrome c kmin., whereas the change in the ubiquinol kmin. was much smaller. From the viscosity studies the extent of diffusional control could be calculated, revealing that the reaction with cytochrome c was mostly diffusion-limited. The viscosity of the membrane was changed by incorporating cholesterol; no significant effect on the ubiquinol kmin. ascribable to diffusion control could be recognized.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Steady-state kinetics of ubiquinol-cytochrome c reductase in bovine heart submitochondrial particles: diffusional effects. 838 78

The nature of the Escherichia coli membrane-bound NADH dehydrogenases and their role in the generation of the proton motive force has been controversial. One E. coli NADH:ubiquinone oxidoreductase has previously been purified to homogeneity, and its corresponding gene (ndh) has been isolated. However, two biochemically distinct E. coli NADH:ubiquinone oxidoreductase activities have been identified by others (K. Matsushita, T. Ohnishi, and H. R. Kaback, Biochemistry 26:7732-7737, 1987). An insertional mutation in the ndh gene has been introduced into the E. coli chromosome, and the resulting strain maintains membrane-bound NADH dehydrogenase activity, demonstrating that a second genetically distinct NADH dehydrogenase must be present. By standard genetic mapping techniques, the map position of a second locus (nuo) involved in the oxidation of NADH has been determined. The enzyme encoded by this locus probably translocates protons across the inner membrane, contributing to the proton motive force.
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PMID:Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli. 838 92

Studies on the effect of various Cd2+ concentrations on substrate oxidation by whole cells of cadmium-sensitive Staphylococcus aureus 17810S showed that oxidation of glutamate or pyruvate was highly sensitive to low Cd2+ concentrations (5 microM), whereas L-lactate oxidation was insensitive even to high Cd2+ concentrations (100 microM). Location of the cadmium-sensitive targets in the enzyme systems involved in oxidation of these substrates was studied in subcellular fractions prepared from cells pretreated with 5 or 100 microM Cd2+. Activities of the cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC)') and pyruvate dehydrogenase complex (PDHC) were strongly inhibited with 5 microM Cd2+, while with 100 microM Cd2+ the inhibition was almost complete. In contrast, activities of the cytoplasmic NAD-dependent glutamate dehydrogenase (NAD-GDH), the membrane-bound NADH dehydrogenase (NDH) and HQNO-sensitive NADH oxidase were not sensitive to 100 microM Cd2+. These data indicate that the accessible, cadmium-sensitive targets are located only in the cytoplasmic ODHC and PDHC. It is postulated that two vicinal dithiols present in ODHC and PDHC may be regarded as the primary cadmium-sensitive targets in the systems oxidizing glutamate or pyruvate. Since activities of the membrane-bound NAD-independent L-lactate dehydrogenase (iLDH) and HQNO-sensitive L-lactate oxidase were not affected by 100 microM Cd2+, this indicates that the L-lactate oxidizing system lacks the accessible, cadmium-sensitive targets. The mechanism of Cd2+ toxicity to energy conservation with glutamate, pyruvate or L-lactate in S. aureus is discussed.
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PMID:Cadmium-sensitive targets in the aerobic respiratory metabolism of Staphylococcus aureus. 895 92

Over 400 P450s have been identified to date in prokaryotes and eukaryotes, plants and animals, mitochondria and endoplasmic reticulum. These enzymes function in areas such as metabolism and steroidogenesis. The eukaryotic members of this gene superfamily of proteins have proved difficult to study because of the hydrophobic nature of their substrates, their various redox partners, and membrane association. To better understand the structure/function relationship of P450s-what determines substrate specificity and selectivity, what determines redox-partner binding, and which regions are involved in membrane binding-we have compared the three crystallized, soluble bacterial P450s (two class I and one class II) and a model of a steroidogenic, eukaryotic P450 (P450arom), to define which structural elements form a conserved structural fold for P450s, what determines specificity of substrate binding and redox-partner binding, and which regions are potentially involved in membrane association. We believe that there is a conserved structural fold for all P450s that can be used to model those P450s that prove intransigent to structural determination. However, although there appears to be a conserved structural core among P450s, there is sufficient sequence variability that no two P450s are structurally identical. NADPH-P450 reductase transfers electrons from NADPH to P450 during the P450 catalytic cycle. This enzyme has usually been thought of as a simple globular protein; however, sequence analysis has shown that NADPH-P450 reductase is related to two separate flavoprotein families, ferredoxin nucleotide reductase (FNR) and flavodoxin. Recent studies by Wolff and his colleagues have shown that the FAD-binding FNR domain and FMN-binding flavodoxin domain of human NADPH-P450 reductase can be independently expressed in Escherichia coli. The subdomains can be used to reconstitute, however poorly, the monooxygenase activity of the P450 system. We have been utilizing the reductase domain of P450BM-3 to study the mechanism of electron transfer from NADPH to P450 in this complex multidomain protein. We have overexpressed both the FNR subdomain and the flavodoxin subdomain in E. coli and fully reconstituted the cytochrome c reductase activity of this enzyme. Our studies have shown that electron transfer from NADPH through the reductase domain to the P450 requires shuttling of the FMN subdomain between the reductase subdomain and the P450. Studies of the factors that control the molecular recognition and interaction among these three proteins are complicated by the weakness of the association and changes in the strength of the interaction depending on the redox state of each of the components. How these structural and mechanistic studies of a soluble bacterial P450 can be extended to gain a better understanding of the control of membrane-bound eukaryotic P450-dependent redox systems is discussed.
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PMID:P450BM-3; a tale of two domains--or is it three? 902 25

Catalysis by microsomal cytochromes P450 requires the membrane-bound enzyme NADPH-cytochrome P450 reductase (P450 reductase), which transfers electrons to the P450 heme via a flavodoxin-like domain. Previously, we reported that Escherichia coli flavodoxin (Fld), a soluble electron transfer protein, directly interacts with bovine cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17) and donates electrons to this enzyme when reconstituted with NADPH-ferredoxin (flavodoxin) reductase (FNR) (Jenkins, C. M., and Waterman, M. R. (1994) J. Biol. Chem. 269, 27401-27408). To investigate whether flavodoxins can serve as useful models of the analogous domain in P450 reductase, we have examined the FNR-Fld system from the cyanobacterium Anabaena. Mutagenesis of two acidic Anabaena Fld residues (D144A and E145A) significantly decreased flavodoxin-supported P450c17 progesterone 17alpha-hydroxylase activity. Specifically, D144A exhibited only 15% of the activity of wild-type Fld, whereas the adjacent mutation, E145A, caused a 40% loss in activity. P450-dependent hydrogen peroxide/superoxide production by wild-type FNR-Fld was measurably higher than that generated by FNR-D144A or FNR-E145A, indicating that the mutations do not lead to P450 heme-mediated electron uncoupling. Interestingly, the D144A and E145A mutants bind with equal or even greater affinity to P450c17 than wild-type Fld. Furthermore, these mutations (D144A and E145A) actually increased cytochrome c reductase activity (35 and 100% higher than wild type). Anabaena Fld residues Asp144 and Glu145 align closely with rat P450 reductase residue Asp208, which has been shown by mutagenesis to be important in electron transfer to P4502B1 but not to cytochrome c (Shen, A. L., and Kasper, C. B. (1995) J. Biol. Chem. 270, 27475-27480). Thus, these residues in flavodoxins and P450 reductase appear to have similar functions in P450 recognition and/or electron transfer, supporting the hypothesis that flavodoxins represent valid models for the FMN-binding domain of P450 reductase.
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PMID:Negatively charged anabaena flavodoxin residues (Asp144 and Glu145) are important for reconstitution of cytochrome P450 17alpha-hydroxylase activity. 927 3

The NADH oxidase of Amphibacillus xylanus shows high NADH-peroxide reductase activity for hydrogen peroxide and alkyl hydroperoxides in the presence of a 22-kDa disulfide-containing protein component (Y. Niimura, L. B. Poole, and V. Massey, J. Biol.Chem. 270, 25645-25650, 1995). It was found that the membrane-bound NADH dehydrogenase of an alkaliphilic Bacillus (YN-1) involved in the respiratory chain also exhibits reductase activity for hydrogen peroxide and cumene hydroperoxide in the presence of the 22-kDa component from Amphibacillus xylanus. Vmax values for these substrates were as high as those of the NADH oxidase of A. xylanus. Although the 38-kDa protein produced by trypsin treatment of NADH dehydrogenase retains NADH dehydrogenase activity, it exhibited no peroxide reductase activity in the presence of the 22-kDa component from A. xylanus. The NADH dehydrogenase of YN-1 might not only catalyze electron flow from NADH to the respiratory chain, but also function for scavenging peroxide.
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PMID:Peroxide reductase activity of NADH dehydrogenase of an alkaliphilic Bacillus in the presence of a 22-kDa protein component from Amphibacillus xylanus. 964 49

The site of metabolism in vascular smooth muscle responsible for the release of nitric oxide (NO) from nitroprusside is not well established. In this study we observed that a membrane-bound NADH oxidoreductase in the pulmonary artery activates nitroprusside to release NO, and we examined whether this process could potentially participate in relaxation to nitroprusside. Relaxation to nitroprusside in bovine calf pulmonary artery is inhibited by a scavenger of NO and by an antagonist of NO stimulation of guanylate cyclase. A flavoprotein probe that inhibits pulmonary artery NADH oxidoreductase (1 micromol/L diphenyliodonium) and electron acceptors for NADH oxidoreductase (0.3 mmol/L nitroblue tetrazolium and 0.1 mmol/L ferricyanide) inhibited pulmonary artery relaxation to nitroprusside, but not to nitroglycerin. Pulmonary arteries were observed to promote the release of NO from nitroprusside in vitro, and NO release was inhibited by the presence of nitroblue tetrazolium, ferricyanide, and diphenyliodonium. In homogenates of pulmonary arteries, NADH (0.1 mmol/L) increased the release of NO from nitroprusside by approximately 6-fold, whereas NADPH, mitochondrial substrates, and other redox cofactors had minimal effects on NO release, and the action of NADH on nitroprusside was inhibited by nitroblue tetrazolium, ferricyanide, and diphenyliodonium. A membrane fraction enriched in NADH oxidoreductase activity showed a NADH-dependent release of NO from nitroprusside; nitroprusside caused NADH consumption, and it also inhibited the NADH-dependent reduction of nitroblue tetrazolium. Thus, a membrane-bound NADH oxidoreductase appears to contribute to the release of NO from nitroprusside, but not nitroglycerin, in calf pulmonary artery.
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PMID:Potential role of a membrane-bound NADH oxidoreductase in nitric oxide release and arterial relaxation to nitroprusside. 993 54

Methanogenic archaea of the order Methanosarcinales which utilize C(1) compounds such as methanol, methylamines or H(2)+CO(2), employ two novel membrane-bound electron transport systems generating an electrochemical proton gradient: the H(2):heterodisulfide oxidoreductase and the F(420)H(2):heterodisulfide oxidoreductase. The systems are composed of the heterodisulfide reductase and either a membrane-bound hydrogenase or a F(420)H(2) dehydrogenase which is functionally homologous to the proton-translocating NADH dehydrogenase. Cytochromes and the novel electron carrier methanophenazine are also involved. In addition, the methyl-H(4)MPT:HS-CoM methyltransferase is bioenergetically relevant. The enzyme couples methyl group transfer with the translocation of sodium ions and seems to be present in all methanogens. The proton-translocating systems with the participation of cytochromes and methanophenazine have been found so far only in the Methanosarcinales.
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PMID:Novel reactions involved in energy conservation by methanogenic archaea. 1047 95

Previous results from our laboratory have shown that NADH-supported electron flow through the Escherichia coli respiratory chain promotes the reduction of cupric ions to Cu(I), which mediates damage of the respiratory system by hydroperoxides. The aim of this work was to characterize the NADH-linked cupric reductase activity from the E. coli respiratory chain. We have used E. coli strains that either overexpress or are deficient in the NADH dehydrogenase-2 (NDH-2) to demonstrate that this membrane-bound protein catalyzes the electron transfer from NADH to Cu(II), but not to Fe(III). We also show that purified NDH-2 exhibits NADH-supported Cu(II) reductase activity in the presence of either FAD or quinone, but is unable to reduce Fe(III). The K(m) values for free Cu(II) were 32 +/- 5 pM in the presence of saturating duroquinone and 22 +/- 2 pM in the presence of saturating FAD. The K(m) values for NADH were 6.9 +/- 1.5 microM and 6.1 +/- 0.7 microM in the presence of duroquinone and FAD, respectively. The quinone-dependent Cu(II) reduction occurred through both O(*-)(2)-mediated and O(*-)(2)-independent pathways, as evidenced by the partial inhibitory effect (30-50%) of superoxide dismutase, by the reaction stoichiometry, and by the enzyme turnover numbers for NADH and Cu(II). The cupric reductase activity of NDH-2 was dependent on thiol groups which were accessible to p-chloromercuribenzoate at low, but not at high, ionic strength of the medium, a fact apparently connected to a conformational change of the protein. To our knowledge, this is the first protein with cupric reductase activity to be isolated and characterized in its biochemical properties.
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PMID:Characterization of an NADH-linked cupric reductase activity from the Escherichia coli respiratory chain. 1051 Feb 71

During respiratory glucose dissimilation, eukaryotes produce cytosolic NADH via glycolysis. This NADH has to be reoxidized outside the mitochondria, because the mitochondrial inner membrane is impermeable to NADH. In Saccharomyces cerevisiae, this may involve external NADH dehydrogenases (Nde1p or Nde2p) and/or a glycerol-3-phosphate shuttle consisting of soluble (Gpd1p or Gpd2p) and membrane-bound (Gut2p) glycerol-3-phosphate dehydrogenases. This study addresses the physiological relevance of these mechanisms and the possible involvement of alternative routes for mitochondrial oxidation of cytosolic NADH. Aerobic, glucose-limited chemostat cultures of a gut2Delta mutant exhibited fully respiratory growth at low specific growth rates. Alcoholic fermentation set in at the same specific growth rate as in wild-type cultures (0.3 h(-1)). Apparently, the glycerol-3-phosphate shuttle is not essential for respiratory glucose dissimilation. An nde1Delta nde2Delta mutant already produced glycerol at specific growth rates of 0.10 h(-1) and above, indicating a requirement for external NADH dehydrogenase to sustain fully respiratory growth. An nde1Delta nde2Delta gut2Delta mutant produced even larger amounts of glycerol at specific growth rates ranging from 0.05 to 0.15 h(-1). Apparently, even at a low glycolytic flux, alternative mechanisms could not fully replace the external NADH dehydrogenases and glycerol-3-phosphate shuttle. However, at low dilution rates, the nde1Delta nde2Delta gut2Delta mutant did not produce ethanol. Since glycerol production could not account for all glycolytic NADH, another NADH-oxidizing system has to be present. Two alternative mechanisms for reoxidizing cytosolic NADH are discussed: (i) cytosolic production of ethanol followed by its intramitochondrial oxidation and (ii) a redox shuttle linking cytosolic NADH oxidation to the internal NADH dehydrogenase.
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PMID:In vivo analysis of the mechanisms for oxidation of cytosolic NADH by Saccharomyces cerevisiae mitochondria. 1078 51


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