Photoaffinity labeling of cytochrome P4501A1 with azidocumene: identification of cumene hydroperoxide binding region

Archives of Biochemistry and Biophysics
T Cvrk, H W Strobel

Abstract

Cumene hydroperoxide can support cytochrome P450-catalyzed reactions in the absence of molecular oxygen, NADPH, and cytochrome P450-NADPH oxidoreductase. Its binding at the cytochrome P450 active site is governed by the structure of the cumene hydroperoxide binding region. In order to define the region of cytochrome P4501A1 at which cumene hydroperoxide binds, we prepared an analog of cumene hydroperoxide for use as a photoaffinity label. p-Azido-isopro-pylbenzene (azidocumene) and its tritiated derivative were photolyzed in water solution by uv light with a half-life of 29 s. The 7-ethoxycoumarin deethylatation catalyzed by P450 using the cumene hydroperoxide-supported system was strongly inhibited by the presence of the label. Covalent binding to the protein after photoactivation was blocked by 50% in the presence of cumene hydroperoxide. HPLC analysis after trypsin digestion of the labeled protein showed that [3H]-azidocumene was attached covalently to the peptide VDMTPAYGLTLK corresponding to residues 492-503 in the 1A1 sequence. The radioactivity level of this fraction was reduced by 50% when the labeling was carried out in the presence of cumene hydroperoxide. To confirm the identified region the labeled protein was cleav...Continue Reading

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Citations

Mar 15, 2003·Xenobiotica; the Fate of Foreign Compounds in Biological Systems·D F V LewisP S Goldfarb
Oct 8, 1999·Archives of Biochemistry and Biophysics·L AntonovicH W Strobel
May 24, 2001·Archives of Biochemistry and Biophysics·T Cvrk, H W Strobel
Jan 16, 2007·Archives of Biochemistry and Biophysics·Samuel L CollomGrover P Miller
Apr 12, 2000·Biochemistry·L K LightningW F Trager

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