Functional importance of a peripheral pocket in mammalian cytochrome P450 2B enzymes

Abstract

The functional importance of a peripheral pocket found in previously published X-ray crystal structures of CYP2B4 and CYP2B6 was probed using a biophysical approach. Introduction of tryptophan within the pocket of CYP2B4 at F202 or I241 leads to marked impairment of 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) or 7-benzyloxyresorufin O-dealkylation efficiency; a similar substitution at F195, near the surface access to the pocket, does not affect these activities. The analogous CYP2B6 F202W mutant is inactive in the 7-EFC O-dealkylation assay. The stoichiometry of 7-EFC deethylation suggested that the decreased activity of F202W and I241W in CYP2B4 and lack of activity of F202W in CYP2B6 coincided with a sharp increase in the flux of reducing equivalents through the oxidase shunt to produce excess water. The results indicate that the chemical identity of residues within this peripheral pocket, but not at the mouth of the pocket, is important in substrate turnover and redox coupling, likely through effects on active site topology.

Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations

“…Introduction of a halogen at the 2-position of the 7-ethoxy side chain of 7-EFC led to a decrease in catalytic efficiency of O -dealkylation by both enzymes and accentuated the positive effects on activity of the amino acid substitutions at positions 194 and 244. Intriguingly, the increases in catalytic efficiency observed with these mutants are diametrically opposite to the detrimental effects of peripheral pocket mutations at residues 202 and 241 and lack of effect of a substitution at position 195 observed previously (26). The substrate preference profiles obtained in this study provide insights into the effects of substrate size and halogen substitution on CYP2B catalysis.…”

“…In this case, the cyclohexyl group of CYMAL-5 bound in the CYP2B4 or 2B6 peripheral pocket would likely prevent changes in the orientation of the I-helix, preventing optimal ligand orientation for oxidation (27). Furthermore, introduction of extra bulk via mutation of select residues to tryptophan severely hampered enzyme activity in both CYP2B4 and 2B6 (26). The snapshots from comparison of the X-ray crystal structures of ligand free CYP2B4 and CYP2B4 F244W and of the (+)-α-pinene bound complexes of CYP2B6 and CYP2B6 Y244W indicated that neither CYMAL-5 nor the distal mutation alters active site topology.…”

“…CYP3A4 was initially crystallized with progesterone bound at a peripheral site, and a recent report utilizing isomeric testosterone dimers found that binding of a monomeric testosterone molecule to a distal allosteric site enhanced binding when the trans -testosterone dimer occupied the enzyme active site (24, 25). In CYP2B enzymes, a peripheral pocket was recently described that is bounded by the E-, F-, G-, and I-helices and is occasionally occupied by 5-cyclohexyl-1-pentyl-β-d-maltoside (CYMAL-5) in X-ray crystal structures (26). This cavity is physically separated from the active site of CYP2B enzymes by the I-helix (Figure 1).…”

“…Previously observed rearrangements of the I-helix likely allow for optimal ligand interaction with the heme group (27). Site-directed mutagenesis of residues within or near the surface of this peripheral pocket revealed that CYP2B4 F202W and CYP2B6 F202W had little or no catalytic activity with the model substrate 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), primarily due to enhanced uncoupling to excess water, whereas CYP2B4 F195W showed no change in 7-EFC O -deethylation activity (26). …”

Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism

“…In addition, site-directed mutagenesis was crucial to understand the structural and functional role of residues in the active site, access channel, and a recently observed peripheral pocket (Kobayashi et al, 1998;Hernandez et al, 2006;Jang et al, 2014Jang et al, , 2015. Our multifaceted approach has revealed how movement of secondary structure elements, such as the F-G helices and I helix, and reorientation of active-site residues, such as F206 and F297, allow CYP2B enzymes to bind compounds as small as a-pinene (molecular weight 5 136 g/mol) and as large as two molecules of amlodipine (molecular weight 5 409 g/mol) with high affinity.…”