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

Abstract

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 16beta- and 16alpha-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.

Activation of the Anticancer Prodrugs Cyclophosphamide and Ifosfamide: Identification of Cytochrome P450 2B Enzymes and Site-Specific Mutants with Improved Enzyme Kinetics

“…We next investigated whether site-directed mutagenesis could be applied to improve the catalytic properties of P450 2B1 toward CPA and IFA, either by increasing 4-hydroxylation or by decreasing metabolism by N-dechloroethylation. We focused our efforts on three 2B1 active site residues, Ile-114 in SRS-1 and Val-363 and Val-367 in SRS-5, based on a large body of earlier studies showing the critical importance of the residues at these positions in dictating substrate specificity differences among P450 2B subfamily members He et al, 1996;Szklarz et al, 1996;Strobel and Halpert, 1997;Domanski et al, 1999). Seven site-specific mutants were prepared and expressed in E. coli.…”

“…Many of these residues belong to five of the six substraterecognition sites (SRSs) for P450 family 2 enzymes that were proposed on the basis of multiple sequence alignments and analogy with P450 101 (Gotoh, 1992). In the case of the P450 2B enzymes, residues 114, 363, and 367 account for many of the functional differences between rat 2B1 and 2B2 (Strobel and Halpert, 1997), rabbit 2B4 and 2B5 Szklarz et al, 1996), rat 2B1 and human 2B6 (Domanski et al, 1999), and rat 2B1 and canine 2B11 . These functional alterations can frequently be explained using three-dimensional homology models based on the structures of bacterial P450s (Szklarz and Halpert, 1997) or P450 2C5 .…”

Identification of 17-α-Ethynylestradiol-Modified Active Site Peptides and Glutathione Conjugates Formed during Metabolism and Inactivation of P450s 2B1 and 2B6

“…information concerning the possible functional role of critical amino acid residues involved in substrate binding and catalysis has come only from homology modeling to crystal structures of P450s (9) and from studies with naturally occurring mutants of P450 enzymes or studies with mutants obtained through site-directed mutagenesis (10,11). An additional important chemical tool for the identification of critical active site amino acid residues involved in the binding and/or catalysis of substrates is the use of mechanism-based inactivators (12).…”

Identification of Amino Acid Residues Involved in the Inactivation of Cytochrome P450 2B1 by Two Acetylenic Compounds: The Role of Three Residues in Nonsubstrate Recognition Sites

“…These amino acids are at residues 303, 360, 363, 367, 473, and 478. The roles of these six residues in substrate specificity, reversible inhibition, and mechanism-based inactivation have been investigated extensively (He et al, 1992(He et al, , 1994Kent et al, 1997;Strobel and Halpert, 1997; Hanna et al, 1998a,b;Lin et al, 2003). Mutating residue 363 in P450 2B2 to the corresponding residue in P450 2B1 resulted in a switch in the metabolism of lidocaine from both N-deethylation and hydroxylation to only N-deethylation (Hanna et al, 1998a).…”

“…Residue 478 is important in the inactivation of P450 2B1 by N-benzyl-1-aminobenzotriazole, steroid metabolism, and the metabolism of several other substrates and inactivators (He et al, 1992;Kent et al, 1997;Strobel and Halpert, 1997;Hanna et al, 1998a).…”