On the substrate specificity of cytochrome P450IIIA1

MJ Namkung, HL Yang, JE Hulla and MR Juchau

Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195.

The instability of the solubilized/purified form, the lack of catalytic activity of the stabilized, macrolide-complexed form, and the compromised catalytic activity of the decomplexed form of steroid- inducible cytochrome P450IIIA1 motivated further investigations of the substrate specificity of this isozyme. A major complementary goal was to identify reactions utilizable as sensitive, specific diagnostic probes for the detection and partial characterization of this isozyme in tissues for which isolation and purification are not practical (e.g., extrahepatic, embryonic tissues, etc.). The approach utilized a combination of a specific, purified inducer, specific inhibitors including triacetyloleandomycin and inhibitory antibodies, and diagnostic probe substrates including the phenoxazone ethers, testosterone, warfarin, 2-acetylaminofluorene, estradiol-17 beta and benzo[a]pyrene. The results obtained indicated that steroid-inducible, rat hepatic P450IIIA1 would catalyze minimal or no O-dealkylation of methoxy-, ethoxy- or pentoxyphenoxazone but catalyzed rapid O- debenzylation of benzyloxyphenoxazone. Hydroxylation of testosterone was specific for the beta face of the molecule at the 2-, 6-, 15- and 16-positions with no detectable conversion to androstenedione and minimal hydroxylation on the alpha face. Both the R- and S-enantiomers of warfarin were attacked at positions 9 and 10, and these reactions appeared to be specific to isozymes of the IIIA family. Aromatic hydroxylation of estradiol-17 beta was efficiently catalyzed, particularly at the 2-position. Hydroxylations of 2-acetylaminofluorene at positions 5 and 7 were catalyzed at relatively rapid rates, but N- hydroxylation of the same substrate was not catalyzed effectively. Hydroxylation of benzo[a]pyrene occurred preferentially at carbon 3 with much lesser activity at carbon 9 and little or no detectable attack at positions 7 or 1. The results indicated that the 2 beta- and 15 beta-hydroxylation of testosterone and the 10-hydroxylation of warfarin would serve as the most useful probes thus far available for detection of the presence of functional P450IIIA1 isozymes in tissues for which isolation and purification are impractical. The results also indicated a very broad, yet selective substrate specificity for the steroid-inducible P450IIIA1.

Volume 34, Issue 5, pp. 628-637, 11/01/1988
Copyright © 1988 by American Society for Pharmacology and Experimental Therapeutics

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