REASSESSMENT OF CYTOCHROME-P450 2B2 - CATALYTIC SPECIFICITY AND IDENTIFICATION OF 4 ACTIVE-SITE RESIDUES

Cytochromes P450 2B metabolize a variety of compounds and have provide d an excellent framework for identifying determinants of substrate spe cificity. 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 2B2(FF) 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 Tie, respectively, at these positions. These enzymes were expressed in Escherichia coli and analyzed with and rostenedione, testosterone, progesterone, ethoxycoumarin, benzyloxyres orufin, and pentoxyresorufin. The expressed 2B2 variants metabolized m ost substrates at rates that were 1-9% of those of 2B1. When steroid r egio- and stereospecificity was examined, the metabolite profiles of e xpressed 2B2 and 2B2(FF) conflicted with the 16 beta- and 16 alpha-hyd roxylation observed for purified hepatic 2B2 from Sprague-Dawley rats. These and other results suggested that the purified hepatic 2B2 conta ined a small percent of the 2B1 enzyme. Masses of tryptic peptides wer e consistent with identity between purified hepatic 2B2 and 2B2(FF). O n the basis of a three-dimensional homology model and the construction and analysis of 2B2 mutants, residues 114, 363, 367, and 478 were ide ntified 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.