NAD(P)H:quinone oxidoreductase (EC 220.127.116.11) (DT-diaphorase) is an FAD- containing enzyme that catalyzes the 2-electron reduction of quinones to hydroquinones using either NADH or NADPH as the electron donor. In this study, FAD was removed by dialyzing the holoprotein against 2 M KBr, and synthetic analogs of FAD were substituted in the flavin binding site as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-mercapto-FAD are stabilized on binding to the enzyme. This is consistent with the fact that the native flavoprotein forms the anion flavin radical upon photoreduction and suggests the presence of a positive charge near the N(1)C(2)O position of the isoalloxazine ring. Reactivity studies using 8-chloro- and 8-mercapto-flavins suggest that the 8 position of the FAD is accessible to the solvent. However, the rates of the reactions were dramatically decreased in the presence of the competitive inhibitor, dicumarol. 6-Mercapto-, 6-thiocyanato-, 6-azido-, and 6-amino-flavins were also used as structural probes. The results indicate that the 6 position is accessible to solvent. Dicumarol binding increases the pK(a) of the enzyme- bound 6-mercapto-flavin from below pH 5.0 to higher than pH 9.0. The results suggest that DT-diaphorase shows the same properties as the C-C transhydrogenases, and the binding of dicumarol elicits a conformational change or an adjustment in the polarity of the FAD pocket. The enzyme reconstituted with oxidized 5-deaza-FAD has significant catalytic activity, confirming that DT-diaphorase is an obligatory 2-electron transfer enzyme and plays a role in the detoxification of quinones and quinoid compounds by reducing them to the relatively stable hydroquinones.
Active site studies of DT-diaphorase employing artificial flavins / G. Tedeschi, S. Chen, V. Massey. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 270:6(1995), pp. 2512-2516.
|Titolo:||Active site studies of DT-diaphorase employing artificial flavins|
TEDESCHI, GABRIELLA (Primo)
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||1995|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1074/jbc.270.6.2512|
|Appare nelle tipologie:||01 - Articolo su periodico|