A new method for preparing flavin-adenine dinucleotide

Whitby, L. G.

doi:10.1042/bj0540437

Cited by 455 publications

(191 citation statements)

References 7 publications

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“…Thereafter, 10% SDS in H 2 O was added to a final concentration of 0.1% and the spectrum of the released FAD was recorded when changes had ceased. An of 11.3 mM -1 cm -1 was used (17) for determination of FAD concentration. The following max values reported earlier (15) were used: hwtMCADH ) 14.8 mM -1 cm -1 ; E376D-MCADH ) 14.8 mM -1 cm -1 ; E376Q-MCADH ) 14.5 mM -1 cm -1 ; E376H-MCADH ) 14.8 mM -1 cm -1 .…”

Section: Materials and Enzymesmentioning

confidence: 99%

Redox Properties of Human Medium-Chain Acyl-CoA Dehydrogenase, Modulation by Charged Active-Site Amino Acid Residues

et al. 1998

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The modulation of the electron-transfer properties of human medium-chain acyl-CoA dehydrogenase (hwtMCADH) has been studied using wild-type and site-directed mutants by determining their midpoint potentials at various pH values and estimating the involved pKs. The mutants used were E376D, in which the negative charge is retained; E376Q, in which one negative charge (pK a ≈ 6.0) is removed from the active center; E99G, in which a different negative charge (pK a ≈ 7.3) also is affected; and E376H (pK a ≈ 9.3) in which a positive charge is present. E m for hwtMCADH at pH 7.6 is -0.114 V. Results for the site-directed mutants indicate that loss of a negative charge in the active site causes a +0.033 V potential shift. This is consistent with the assumption that electrostatic interactions (as in the case of flavodoxins) and specific charges are important in the modulation of the electron-transfer properties of this class of dehydrogenases. Specifically, these charge interactions appear to correlate with the positive E m shift observed upon binding of substrate/product couple to MCADH [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990) Biochemistry 29, 3709-3715], which coincides with a pK increase of Glu376-COOH from ∼6 to 8-9 [Rudik, I., Ghisla, S., and Thorpe, C. (1998) Biochemistry 37, 8437-8445]. From the pH dependence of the midpoint potentials of hwtMCADH two mechanistically important ionizations are estimated. The pK a value of ∼6.0 is assigned to the catalytic base, Glu376-COOH, in the oxidized enzyme based on comparison with the pH behavior of the E376H mutant, it thus coincides with the pK value recently estimated [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The pK a of ∼7.1 is assigned to Glu376-COOH in reduced hwtMCADH. Comparable values for these pK a s for Glu376-COOH in pig kidney MCADH are pK ox ) 6.5 and pK red ) 7.9. The E m measured for K304E-MCADH (a major mutant resulting in a deficiency syndrome) is essentially identical to that of hwtMCADH, indicating that the disordered enzyme has an intact active site.Mitochondrial fatty acid oxidation is a major energy source for mammals (1). The initial and key enzymes in -oxidation are the acyl-CoA dehydrogenases, members of a broad family, which catalyze the dehydrogenation of fatty acids with chain lengths of 4 up to >18 carbon atoms. Mediumchain acyl-CoA dehydrogenase (MCADH), with an optimum activity for C 8 -substrates, is the best studied member of this family. The reaction it catalyzes occurs formally in two steps as shown in eqs 1 and 2 (2, 3):where E ) MCADH, SH 2 ) reduced substrate, P ) product, ETF ) electron transferring flavoprotein, and its (•) semiquinone form. 1 The reductive half-reaction proceeds via the abstraction of both a proton and a hydride ion from the R-and -carbons of the thioester-CoA substrate, respectively, (4). Glu376-COO -is the proton abstracting base (5, 6). The carbonyl oxygen of the thioester is involved in two essential hydrogen bonds, one to the ribityl 2-hydroxyl group...

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Section: Materials and Enzymesmentioning

confidence: 99%

Redox Properties of Human Medium-Chain Acyl-CoA Dehydrogenase, Modulation by Charged Active-Site Amino Acid Residues

et al. 1998

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“…Similar results were obtained by incubating the enzyme for 30 min in 7 M urea or in 80% methanol at 75°C. The released cofactor was identified as FAD by HPLC; based upon the ⑀ 450 value for FAD of 11.3 mM Ϫ1 cm Ϫ1 (23), an extinction coefficient of 7.4 mM Ϫ1 cm Ϫ1 was determined at 336 nm for the enzyme-bound flavin. A stoichiometry of 0.98 FAD/monomer could be calculated from these results.…”

Section: Spectralmentioning

confidence: 99%

“…After incubation of the free flavin at pH 7.1, a single species eluting at 18.38 min was obtained. Based upon the known ⑀ 450 value for FAD (23), an extinction coefficient of 10.7 mM Ϫ1 cm Ϫ1 was calculated at 346 nm for the flavin extracted at pH 2.1.…”

Section: Fig 2 Circular Dichroism Spectrum Of Nitroalkane Oxidasementioning

confidence: 99%

Identification of the Naturally Occurring Flavin of Nitroalkane Oxidase from Fusarium oxysporum as a 5-Nitrobutyl-FAD and Conversion of the Enzyme to the Active FAD-containing Form

Gadda

Edmondson

Russell

et al. 1997

Journal of Biological Chemistry

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Nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to aldehydes with production of nitrite and hydrogen peroxide. The UVvisible absorbance spectrum of the purified enzyme shows a single absorption peak at 336 nm with an extinction coefficient of 7.4 mM ؊1 cm ؊1 . Upon denaturation of the enzyme at pH 7.0, a stoichiometric amount of FAD is released. The spectral properties of the enzyme as isolated are consistent with an N(5) adduct of the flavin. This is not due to a covalent linkage with the protein, since the free flavin adduct can be isolated from the enzyme at pH 2.1. The free flavin adduct shows an absorbance spectrum with a max at 346 nm (10.7 mM ؊1 cm ؊1 ) and is not fluorescent. Under alkaline conditions the free adduct decays, yielding FAD; the rate of this process is pH-dependent with a pK a of 7.4. Adduct decay is also observed with the native enzyme; in this case, however, the rate of decay is 160-fold slower (at pH 8.0) and not dependent on pH. During this process a large increase in enzymatic activity (ϳ26-fold at pH 7.0) is observed, the rate of which is equal to the rate of flavin adduct conversion to FAD. Thus, the native flavin adduct is not active but can be converted to FAD, the active form of the flavin. Maximal activation is pH-and FAD-dependent; two groups with pK a values of 5.65 ؎ 0.25 and 8.75 ؎ 0.05 must be unprotonated and protonated, respectively. The m/z ؊ of the free flavin adduct is 103.0645 higher than that of FAD, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This corresponds to a molecule of nitrobutane linked to FAD. A mechanism is proposed for the formation in vivo of the nitrobutyl-FAD of nitroalkane oxidase.Flavoprotein oxidases comprise a large group of enzymes that catalyze the removal of a hydride equivalent from a substrate, transferring the electrons initially to the flavin cofactor and then to molecular oxygen to form hydrogen peroxide and the oxidized product. The most studied examples are the ␣-hydroxy acid oxidases, such as lactate oxidase (1), glycolate oxidase (2), and flavocytochrome b 2 (3) and the amino acid oxidases, of which D-amino acid oxidase is the best understood (4). There is strong evidence that the first step in the formation of the respective keto or imino acid products is removal of the substrate ␣-proton to form a carbanion. This conclusion is based upon halide elimination from ␤-substituted substrates (5), mechanism-based inactivation by propargyllic substrates (6), and kinetic isotope effects (7). The substrate carbanion is proposed to form an adduct at the N(5) position of flavin; such an adduct would then break down to form the reduced flavin and keto or imino acid product (8). Evidence for such an adduct comes from: (a) the trapping of a species with the properties of an N(5) adduct when glycolate is a substrate for lactate oxidase (9) and (b) the formation of adducts when nitromethane or nitroethane carbanion is used as substrate for D-amino acid oxidase (10). ...

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“…Concentration of FMN was estimated spectrophotometrically by using £450 = 12.2 mM -I cm -1 (ref. 13). PNP was estimated by absorption maximum (£253=3.7 mM -I cm -I ) in 0.1 M phosphate buffer (pH 7.0).14)…”

mentioning

confidence: 99%

Molecular and Enzymatic Properties of Pyridoxamine (Pyridoxine) 5′-Phosphate Oxidase from Baker’s Yeast

Tsuge

Ozeki

Ohashi

1980

Agricultural and Biological Chemistry

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A new method for preparing flavin-adenine dinucleotide

Cited by 455 publications

References 7 publications

Redox Properties of Human Medium-Chain Acyl-CoA Dehydrogenase, Modulation by Charged Active-Site Amino Acid Residues

Redox Properties of Human Medium-Chain Acyl-CoA Dehydrogenase, Modulation by Charged Active-Site Amino Acid Residues

Identification of the Naturally Occurring Flavin of Nitroalkane Oxidase from Fusarium oxysporum as a 5-Nitrobutyl-FAD and Conversion of the Enzyme to the Active FAD-containing Form

Molecular and Enzymatic Properties of Pyridoxamine (Pyridoxine) 5′-Phosphate Oxidase from Baker’s Yeast

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