Formation of a novel reversible cytochrome P450 spectral intermediate: role of threonine 303 in P450 2E1 inactivation

Biochemistry
Anna L BlobaumPaul F Hollenberg

Abstract

tert-Butyl acetylene (tBA) is a mechanism-based inactivator of cytochromes P450 2E1 and 2E1 T303A; however, the inactivation of the T303A mutant could be reversed by overnight dialysis. The inactivation of P450 2E1 T303A, but not the wild-type 2E1 enzyme, by tBA resulted in the formation of a novel reversible acetylene-iron spectral intermediate with an absorption maximum at 485 nm. The formation of this intermediate required oxygen and could be monitored spectrally with time. Although the alternate oxidants tert-butyl hydroperoxide (tBHP) and cumene hydroperoxide (CHP) supported the inactivation of wild-type P450 2E1 by tBA in a reductase- and NADPH-free system, only tBHP supported the inactivation of the 2E1 T303A mutant. The losses in enzymatic activity occurred concomitantly with losses in the native P450 heme, which were accompanied by the formation of tBA-adducted heme products. The inactivations supported by tBHP and CHP were completely irreversible with overnight dialysis. Spectral binding constants (K(s)) for the binding of tBA to the 2E1 P450s together with models of the enzymes with the acetylenic inactivator bound in the active site suggest that the T303A mutation results in increased hydrophobic interactions betwee...Continue Reading

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Citations

Jan 3, 2016·Biochimica Et Biophysica Acta·Arthur G RobertsRheem A Totah
Dec 25, 2007·Chembiochem : a European Journal of Chemical Biology·Kersten S RabeChristof M Niemeyer
Jan 13, 2006·Journal of Inorganic Biochemistry·Jonathan P ClarkStephen K Chapman
Nov 5, 2005·Biochemical and Biophysical Research Communications·Peter H J KeizersJan N M Commandeur
Sep 15, 2005·Biochemical and Biophysical Research Communications·Chitra SridarPaul F Hollenberg
Jun 20, 2017·Biochimica Et Biophysica Acta. Proteins and Proteomics·Julie Ducharme, Karine Auclair
Apr 14, 2006·The Journal of Pharmacology and Experimental Therapeutics·Namandjé N BumpusPaul F Hollenberg
Dec 22, 2005·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Anna L Blobaum
Jun 18, 2019·Drug Metabolism Reviews·Paul R Ortiz de Montellano

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