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Reassessment of Cytochrome P450 2B2:  Catalytic Specificity and Identification of Four Active Site Residues

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Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721
Cite this: Biochemistry 1997, 36, 39, 11697–11706
Publication Date (Web):September 30, 1997
Copyright © 1997 American Chemical Society

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    Cytochromes P450 2B metabolize a variety of compounds and have provided an excellent framework for identifying determinants of substrate specificity. Among the rat 2B enzymes, a puzzling difference has emerged between the reported substrate specificity of purified hepatic 2B2 and that of certain 2B1 mutants containing 2B1 → 2B2 substitutions. To address these discrepancies, we have characterized two 2B2 variants. A cDNA clone designated 2B2FF was obtained from phenobarbital-induced Lewis rats and, like some previously isolated variants, was found to contain phenylalanine at positions 58 and 114. A second 2B2 clone was generated by restoring Leu and Ile, respectively, at these positions. These enzymes were expressed in Escherichia coli and analyzed with androstenedione, testosterone, progesterone, ethoxycoumarin, benzyloxyresorufin, and pentoxyresorufin. The expressed 2B2 variants metabolized most substrates at rates that were 1−9% of those of 2B1. When steroid regio- and stereospecificity was examined, the metabolite profiles of expressed 2B2 and 2B2FF conflicted with the 16β- and 16α-hydroxylation observed for purified hepatic 2B2 from Sprague-Dawley rats. These and other results suggested that the purified hepatic 2B2 contained a small percent of the 2B1 enzyme. Masses of tryptic peptides were consistent with identity between purified hepatic 2B2 and 2B2FF. On the basis of a three-dimensional homology model and the construction and analysis of 2B2 mutants, residues 114, 363, 367, and 478 were identified as determinants of substrate specificity. In addition, 2B1 and the expressed 2B2 variants showed differential susceptibility to the mechanism-based inactivators chloramphenicol and N-(2-p-nitrophenethyl)chlorofluoroacetamide.

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     Supported by Grant ES03619 and Center Grant ES06694 from the National Institutes of Health. Presented in part at the American Society for Pharmacology and Experimental Therapeutics Annual Meeting in San Diego, CA, March 1997.


     To whom correspondence and reprint requests should be addressed. Phone:  (520) 626-4489. Fax:  (520) 626-2466. E-mail:  strobel@

     Abstract published in Advance ACS Abstracts, September 15, 1997.

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