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

The reduction of the azo dye, amaranth, by rat liver microsomes is inhibited about 90% by carbon monoxide, suggesting that the reaction largely depends on cytochrome P-450. Reducing equivalents for this reaction are supplied by NADPH. This reaction is stimulated by riboflavin, FMN and FAD, as well as by methylviologen. A large fraction of the stimulated reaction is not blocked by CO, indicating that there is a pathway of electron transfer which is independent of cytochrome P-450. Rat liver microsomes can reduce FAD, with reducing equivalents supplied by NADPH. The FADH2 thus produced is quickly oxidized by amaranth so that two FADH2 are oxidized for every amaranth reduced. The same stoichiometry is observed with photochemically prepared FADH2, formed in the absence of microsomes.
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PMID:The stimulation of microsomal azoreduction by flavins. 715 Jun 36

Lipoamide dehydrogenase from Escherichia coli, a dimeric flavoprotein in the pyridine nucleotide-disulfide oxidoreductase family of enzymes, catalyzes the reduction of NAD+ by dihydrolipoamide. The two electrons are transferred via a redox active disulfide and FAD. Cys44 and Cys49 comprise the redox active disulfide, Cys44 interchanging with dihydrolipoamide and Cys49 interacting with the flavin. Each of these residues has been mutated to serine (C44S, C49S). The altered enzymes showed minute amounts of activity, 0.003% for C44S and 0.012% for C49S using the physiological substrates dihydrolipoamide and NAD+. These very low activities were expected, since the disulfide was no longer present in C44S and C49S, making dithiol-disulfide interchange impossible. However, the enzymes were capable of catalyzing reactions using NADH as the electron donor and alternate electron acceptors: K3Fe(CN)6, thio-NAD+, DCIP, and O2. These activities with NADH indicated that interaction of C44S and C49S with pyridine nucleotides was not affected greatly by the mutation. The pH dependence of the charge-transfer absorbance of C44S gives pKa values of 2.7, associated with titration of Cys49, and 9.5, associated with titration of the acid-base catalyst, His444'. A pKa of 5.1 was estimated for Cys44 in C49S from the pH dependence of its reactivity with methyl methanethiosulfonate. The fluorescence of the FAD in oxidized wild type lipoamide dehydrogenase is markedly temperature dependent, while the remaining fluorescence of two-electron-reduced enzyme is independent of temperature. The fluorescence of the FAD in C44S and in C49S is likewise independent of temperature. The FAD of C44S and C49S is stoichiometrically titrated by 1 equiv of sodium dithionite. However, the FAD of C44S is markedly less completely reduced by 1 equiv of NADH than is the FAD of C49S. Ferricyanide stoichiometrically reoxidizes the FADH2 of both altered forms of the enzyme.
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PMID:Characterization of lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S. 754 8

The cysteines that comprise the active site disulfide in lipoamide dehydrogenase have been individually mutated to a serine residue to give the altered enzymes, C44S and C49S, making it possible to study the redox behavior of the FAD in the absence of the disulfide. The redox potential of the FAD in C44S and C49S was -379 and -345 mV, respectively, at pH 7.0, 25 degrees C. A plot of the redox potential as a function of pH for C49S gave slopes of 57 mV/pH from pH 5.0 to 7.9 and 10 mV/pH from pH 7.9 to 8.8. The plot of the redox potential as a function of pH for C44S gave slopes of 70 mV/pH from pH 5.0 to 7.9 and 4 mV/pH from pH 7.9 to 8.38. The change in the slope at pH 7.9 is associated with the ionization (pKa) of the FADH2 to FADH- in the reduced form of both enzymes. These determinations show that the redox potential of the FAD in C49S, in C44S, and in wild type enzyme is modulated by the electronegativity of its nearest neighbor, hydroxyl, thiolate, or disulfide, and that the flavin is bound more tightly to the oxidized forms of these enzymes than to the reduced forms. The redox potentials of these enzymes determined using NADH and NADPH at pH 7.6, 25 degrees C are as follows: C44S, -350 mV, -369 mV; C49S, -328 mV, -353 mV, respectively. Thus, pyridine nucleotide binding raises the redox potential of the flavin, showing that both substrates bind more tightly to the reduced form of the enzymes, as well as tighter binding of NADH to the enzymes than that of NADPH. Kd values for the binding of NADH and NADPH to oxidized C44S and C49S were determined in pre-steady-state kinetics at pH 7.6 and 25 degrees C, which were monophasic when NADPH was the reductant and biphasic with NADH. The binding constants for NADPH were 660 microM for C44S and 500 microM for C49S; using NADH, the binding constants were 137 microM for C44S and 23 microM for C49S. Fluorescence and absorbance spectrophotometry were used to determine the binding of NAD+ to the oxidized forms of the enzymes as 275 microM and 270 microM for C44S and C49S, respectively.
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PMID:Lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S. Redox properties of the FAD and interactions with pyridine nucleotides. 754 9

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.
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PMID:The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction. 756 29

Trypanothione reductase was purified to homogeneity from Leishmania donovani promastigotes transfected with the expression plasmid pTEX-LdTR. The physical, spectral and kinetic properties were found to be similar to those obtained from other pathogenic trypanosomatids. The substrates trypanothione disulfide and NADPH exhibit Michaelis-Menten saturation kinetics with Km values of 36 microM and 9 microM, respectively, the former yielding a kcat/Km of 5.0 x 10(6) M-1 s-1. Like other trypanothione reductases, the leishmania enzyme is unable to use glutathione disulfide as substrate. Both trypanothione reductase and the analogous mammalian enzyme, glutathione reductase, are inhibited by trivalent but not pentavalent anti-leishmanial antimonials. Inhibition by trivalent sodium antimonyl gluconate (Triostam) occurs in a time-dependent manner, with the pseudo-first-order rate constants of inhibition being linearly related to drug concentration. Inhibition proceeds until an apparent equilibrium between active enzyme/free drug and inactive enzyme-drug complex is reached. MelT, an adduct of melarsen oxide and dihydrotrypanothione which is a competitive inhibitor of the disulfide binding site of trypanothione reductase, confers protection against Triostam. Prior reduction of the catalytically active disulfide bridge by NADPH is essential for inhibition. Spectral analysis shows that the broad absorbance band centred on 530 nm, characteristic of the charge-transfer complex in the two-electron-reduced EH2 enzyme, is lost upon addition of Triostam. Further spectral changes resemble those associated with reduction of the FAD prosthetic group to FADH2. Inhibition by Triostam is readily reversed by dilution or addition of the dithiols 2,3-dimercaptopropanol, 2,3-dimercaptosuccinate or dithiothreitol, but not dihydrotrypanothione, suggesting that this trypanosomatid-unique metabolite is unlikely to protect the enzyme from inhibition in whole cells. A mechanism consistent with these observations is proposed.
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PMID:Trypanothione reductase from Leishmania donovani. Purification, characterisation and inhibition by trivalent antimonials. 760 16

The kinetic mechanism of the flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase with its natural substrates 2-aminobenzoyl-CoA, NADH and O2 has been investigated using the stopped-flow technique. Initial rate measurements indicate the formation of a ternary complex between oxidized enzyme and the two substrates 2-aminobenzoyl-CoA and NADH, a turnover number of approximately 40 min-1 was found at pH 7.4 and 4 degrees C. 2-Aminobenzoyl-CoA binds to oxidized enzyme to form a complex which is in a approximately 1:1 equilibrium with a second, spectrophotometrically distinguishable one. Binding of 2-amino benzoyl-CoA to reduced enzyme is, in contrast, a simple second-order process. Reduction of oxidized enzyme, both uncomplexed and in complex with 2-aminobenzoyl-CoA, by NADH is strongly biphasic. The first fast phase yields enzyme in which 50% of the total FAD is reduced to the FADH2 state. This rate is not affected by the presence of 2-aminobenzoyl-CoA. In contrast, 2-aminobenzoyl-CoA enhances approximately 100-fold the second phase, the reduction of the residual 50% FAD. This second phase of reduction (kobs = 2.0 s-1) is partially rate-limiting in catalysis. The oxygen reaction of uncomplexed, reduced enzyme is also biphasic and no oxygenated intermediate was detected. Reoxidation of substrate-complexed, reduced enzyme involves three spectroscopically distinguishable species. The first observable intermediate is highly fluorescent suggesting that it consists largely of flavin-4a-hydroxide. Thus, insertion of oxygen into 2-aminobenzoyl-CoA is essentially complete at this point and has a kobs > or = 80 s-1. The subsequent phase is accompanied by formation of the main product, 2-amino-5-oxocyclohex-1-enecarboxyl CoA. This step consists in a hydrogenation of the primary, oxygenated and non-aromatic CoA intermediate; it has a rate approximately 1.3 s-1, which is thus the second rate-limiting step in catalysis. As a side reaction of the oxidized enzyme and at low NADH concentrations the initially formed product disappears at a very slow rate (kobs approximately 0.05 s-1). This third 'post-catalytic' process is not relevant for catalysis. The primary product 2-amino-5-oxocyclohex-1-enecarboxyl-CoA is dehydrogenated by the oxidized enzyme to yield the aromatic 2-amino-5-hydroxybenzoyl-CoA as secondary product. The reduced enzyme formed in this process is reoxidized by O2 to form H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Kinetic and mechanistic studies on the reactions of 2-aminobenzoyl-CoA monooxygenase/reductase. 760 43

In a previous paper, we demonstrated that the reductive half-reaction of medium-chain fatty acyl-CoA dehydrogenase (MCAD), utilizing octanoyl-CoA as physiological substrate, generates two (kinetically distinct) forms of the reduced enzyme (MCAD-FADH2) - octenoyl-CoA charge-transfer complexes [Kumar, N.R., & Srivastava, D.K. (1994) Biochemistry 33, 8833-8841]. We present evidence that octenoyl-CoA dissociates from the second (most stable) charge-transfer complex (referred to as CT2) via two alternative ("facile" and "restricted") pathways. The dissociation of octenoyl-CoA via the facile pathway involves the reversal of the overall reductive half-reaction of the enzyme, generating MCAD-FAD - octanoyl-CoA as the Michaelis complex, followed by dissociation of the latter complex into MCAD-FAD + octanoyl-CoA. Hence, via this pathway, octenoyl-CoA is released from the enzyme site in the form of octanoyl-CoA. In contrast, the restricted pathway involves a direct (albeit slow) dissociation of octenoyl-CoA from CT2 to yield MCAD-FADH2 + octenoyl-CoA. The kinetic profile for the dissociation of octenoyl-CoA via the restricted pathway matches the rate of oxidation of the reduced flavin (within CT2) by O2. This suggests that the oxidase activity of the enzyme remains suppressed as long as the reduced enzyme predominates in the form of the charge-transfer complex(es). The oxidase activity of the enzyme emerges concomitantly with the conversion of CT2 to the MCAD-FADH2 - octenoyl-CoA Michaelis complex. The energetic basis for the dissociation of octenoyl-CoA via the facile and restricted pathways and the mechanism of suppression of the oxidase activity of the enzyme are discussed.
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PMID:Facile and restricted pathways for the dissociation of octenoyl-CoA from the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-FADH2-octenoyl-CoA charge-transfer complex: energetics and mechanism of suppression of the enzyme's oxidase activity. 762 13

A flavoprotein from Amphibacillus xylanus catalyzes the reduction of oxygen to hydrogen peroxide. Each polypeptide chain in the tetrameric enzyme contains 5 cysteine residues. The complete reduction of enzyme by dithionite requires 6 electrons. Such behavior indicates the presence of redox centers in addition to the FAD, and these could be disulfides. In order to assess the catalytic role of disulfide in the enzyme, 2 of the cysteines (Cys-337 and Cys-340), which show a high degree of homology with alkyl hydroperoxide reductase F52a protein and thioredoxin reductase, have been changed to serines by site-directed mutagenesis of the cloned flavoprotein gene (individually and in a double mutant). Titration of the three mutant enzymes, lacking Cys-337, Cys-340, or both cysteines, requires only 2 electron eq to reach the reduced flavin state. These results indicate the absence of a redox-active disulfide and demonstrate the involvement of Cys-337 and Cys-340 in the redox-active disulfide. The catalytic activity of the three enzymes was examined by steady-state analysis. The Km for NADH and oxygen and the kcat value of these mutant enzymes were essentially the same as those of wild type. The NADH oxidase activities were also accelerated markedly in the presence of free FAD, which is the case for wild-type enzyme. The NADH:5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) oxidoreductase activities of all mutant enzymes were less than 3% of the activity of wild-type enzyme. The weak DTNB reductase activities in the mutant enzymes lacking Cys-337 or Cys-340 may occur through direct reduction of the mixed disulfide Cys-337-thiol or Cys-340-thiol and nitrothiobenzoate by FADH2. However, the weak DTNB reductase activity in the mutant enzyme lacking both cysteines indicates that FADH2 can reduce either DTNB or another disulfide directly, albeit inefficiently. These results suggest intramolecular dithiol-disulfide interchange reactions in the flavoprotein.
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PMID:Role of cysteine 337 and cysteine 340 in flavoprotein that functions as NADH oxidase from Amphibacillus xylanus studied by site-directed mutagenesis. 772 98

The flavoprotein NADH peroxidase from Enterococcus faecalis 10C1 has been shown to contain, in addition to FAD, an unusual cysteine-sulfenic acid (Cys-SOH) redox center. The non-flavin center cycles between reduced (Cys-SH) and oxidized (Cys-SOH) states, and the 2.16 A crystal structure of the non-native cysteine-sulfonic acid (Cys-SO3H) form of the wild-type peroxidase supports the proposed catalytic role of Cys42. In this study, we have employed a site-directed mutagenesis approach in which Cys42 is replaced with Ser and Ala, neither side chain of which is capable of redox activity. Reductive titrations of both C42S and C42A mutants lead directly to full FAD reduction with 1 equiv of either dithionite or NADH, consistent with elimination of the Cys-SOH center. Direct determinations of the redox potentials for the FAD/FADH2 couples yield values of -219 and -197 mV, respectively, for C42S and C42A peroxidases, indicating that the presence of Cys42-SH in the two-electron-reduced wild-type enzyme lowers the flavin potential by approximately 100 mV. Anaerobic stopped-flow analyses of the reduction of C42S and C42A peroxidases by NADH demonstrate that in both cases flavin reduction is rapid; these results are confirmed by enzyme-monitored, steady-state kinetic analyses which, in addition, give turnover numbers approximately 0.04% that of wild-type enzyme. These results are entirely consistent with the role proposed for Cys42 in the catalytic redox cycle of wild-type NADH peroxidase and indirectly support its function as a peroxidatic center in the homologous NADH oxidase.
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PMID:Analysis of the kinetic and redox properties of NADH peroxidase C42S and C42A mutants lacking the cysteine-sulfenic acid redox center. 781 35

Cytochrome P450 102 is a catalytically self-sufficient monooxygenase isolated from barbiturate-induced Bacillus megaterium. The enzyme contains FAD, FMN, and heme in a single polypeptide chain of 1048 residues, and each of the cofactors is believed to be located in a separate domain. In the present study we have used exhaustive endogenous proteolysis to produce a 45 kDa fragment of the cytochrome. This fragment bound the 2',5'-adenosine diphosphate moiety of NADP(H) strongly, with approximately the same dissociation constant as in the native enzyme, and contained only FAD (0.93 equivalents per polypeptide, epsilon 453nm = 11,200 M-1cm-1). Reduction of the flavin by sodium dithionite proceeded quite slowly to yield FADH2, but no stable semiquinone species was produced upon air re-oxidation. In contrast, NADPH rapidly reduced this FAD/NADP(H) domain aerobically to produce the FADH. semiquinone radical. At a 75:1 molar ratio of the FAD/NADP(H) domain to the P450 102 heme domain, no laurate hydroxylase activity was observed. Gas-phase sequence analysis showed the presence of two major sequences beginning at Phe646 (403 residues, MW 45,033) and Asp652 (397 residues). These data are in agreement with the crystal structures of related enzymes and closely define the boundary of the FAD/NADP+ domain in P450 102.
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PMID:On the domain structure of cytochrome P450 102 (BM-3): isolation and properties of a 45-kDa FAD/NADP domain. 807 51


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