@article{KrauthSiegel1985analo-7539,
  year={1985},
  title={FAD analogues as prosthetic groups of human glutathione reductase : properties of the modified enzyme species and comparisons with the active site structure},
  number={2},
  volume={148},
  journal={European Journal of Biochemistry},
  pages={335--344},
  author={Krauth-Siegel, Renate Luise and Schirmer, Ralf H. and Ghisla, Sandro}
}

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    <dcterms:issued>1985</dcterms:issued>
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    <dc:creator>Ghisla, Sandro</dc:creator>
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    <dc:contributor>Krauth-Siegel, Renate Luise</dc:contributor>
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    <dcterms:bibliographicCitation>First publ. in: European Journal of Biochemistry 148 (1985), 2, pp. 335-344</dcterms:bibliographicCitation>
    <dc:creator>Krauth-Siegel, Renate Luise</dc:creator>
    <dc:language>eng</dc:language>
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    <dc:contributor>Ghisla, Sandro</dc:contributor>
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:35:14Z</dc:date>
    <dcterms:title>FAD analogues as prosthetic groups of human glutathione reductase : properties of the modified enzyme species and comparisons with the active site structure</dcterms:title>
    <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/7539/1/Eur_J_Biochem_1985_Krauth_SiegelFAD_analogues_as_prosthetic_grou.pdf"/>
    <dcterms:abstract xml:lang="eng">1. Human glutathione reductase (NADPH + GSSG + H+ {rightleftharpoons} NADP+ + 2 GSH) is a suitable enzyme for correlating spectroscopic properties and chemical reactivities of protein-bound FAD analogues with structural data. FAD, the prosthetic group of the enzyme, was replaced by FAD analogues, which were modified at the positions 8, 1, 2, 4, 5 and 6, respectively, of the isoalloxazine ring.&lt;br /&gt;2. When compared with a value of 100% for native glutathione reductase, the specific activities of most enzyme species ranged from 40% to 17%, in the order of the prosthetic groups 8-mercapto-FAD &gt; 8-azido-FAD = 8-F-FAD = 8-Cl-FAD &gt; 4-thio-FAD = 1-deaza-FAD &gt; 2-thio-FAD. The enzymic activities indicate a correct orientation of the bound analogues. The enzyme species containing 5-deaza-FAD and 6-OH-FAD, respectively, had no more glutathione reductase activity than the FAD-free apoenzyme. 5-Deaza-FAD glutathione reductase was crystallized for X-ray diffraction analysis.&lt;br /&gt;3. Detailed studies were focussed on position 8 of the flavin. 8-Cl-FAD · glutathione reductase and 8-F-FAD · glutathione reductase reacted only poorly with HS− to give 8-mercapto-FAD · glutathione reductase, which suggests that the region around Val61 hinders the halogen anion from leaving the tetrahedral intermediate. Other experiments showed that position 8 is accessible to certain solvent-borne reagents. 8-Mercapto-FAD · glutathione reductase, for instance, reacted readily and stoichiometrically with the thiol reagent methylmethanethiosulfonate.&lt;br /&gt;4. 8-Mercapto-FAD · glutathione reductase does not exhibit a long wavelength charge transfer absorption band upon reduction, as it is the case for the 2-electron-reduced FAD-containing enzyme. This behaviour indicates that the charge transfer interaction between flavin and the thiolate of Cys63 in the native enzyme is not per se essential for catalysis. The absorption spectrum of the blue anionic 8-mercapto-FAD bound to glutathione reductase suggests that the protein concurs to the stabilization of a negative charge in the pyrimidine subnucleus. In light of the protein structure this effect is attributed to the dipole moment of α-helix 338 354 which starts out close to the N(1)/C(2)/O(2α) region of the flavin.&lt;br /&gt;5. 1-Deaza-FAD binds as tightly as FAD to the apoenzyme. The resulting holoenzyme was found to be enzymically active but structurally unstable. In this respect 1-deaza-FAD · glutathione reductase mimics the properties of the enzyme species found in inborn glutathione reductase deficiency. The high specific activity of 1-deaza-FAD · glutathione reductase shows that the hydrogen bond between N(1) and Thr339 in the native enzyme is not essential for catalysis.&lt;br /&gt;6. 2-Thio-FAD and 4-thio-FAD were used as probes in order to confirm the crystallographic result that the region around N(1)/O(2α)/N(3) and around O(4α) of the native enzyme are practically inaccessible to solventborne reagents. Reaction of 4-thio-FAD · glutathione reductase with a large excess of H2O2 very slowly led to a catalytically inactive protein which may contain covalently bound FAD. In contrast to the native FAD enzyme, 2-electron-reduced 4-thio-FAD · glutathione reductase contains reduced flavin whereas the redox-active pair Cys58-Cys63 is likely to be present in the disulfide form. As 4-thio-FAD is a catalytically competent prosthetic group, this means that the reducing equivalents can flow from NADPH via the two redox systems of the enzyme to GSSG even when the flavin analogue has a less negative redox potential than the subsequent dithiol/disulfide group.&lt;br /&gt;7. 6-OH-FAD · Glutathione reductase showed no detectable reductase activity. The pK value of 7.2 ± 0.3 found for protein-bound 6-OH-FAD is the same as for free 6-OH-FAD. The interpretation that 6-OH-FAD binds poorly to apoglutathione reductase is consistent with the structure of the native enzyme. Three amino acid residues, namely Gly62, Cys63 and Lys66, are in van der Waals contact with C(6) and do not allow the introduction of a bulky OH-group at this position. The discrimination against 6-OH-FAD which occurs beside FAD in vivo might be the result of a selection process.</dcterms:abstract>
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    <dc:contributor>Schirmer, Ralf H.</dc:contributor>
    <dc:creator>Schirmer, Ralf H.</dc:creator>
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