Inhibition of CYP2B4 by 2-ethynylnaphthalene: evidence for the co-binding of substrate and inhibitor within the active site.

2-ethynylnaphthalene (2EN) is an effective mechanism-based inhibitor of CYP2B4. There are two inhibitory components: (1) irreversible inactivation of CYP2B4 (a typical time-dependent inactivation), and (2) a reversible component. The reversible component was unusual in that the degree of inhibition was not simply a characteristic of the enzyme-inhibitor interaction, but dependent on the size of the substrate molecule used to monitor residual activity. The effect of 2EN on the metabolism of seven CYP2B4 substrates showed that it was not an effective reversible inhibitor of substrates containing a single aromatic ring; substrates with two fused rings were competitively inhibited by 2EN; and larger substrates were non-competitively inhibited. Energy-based docking studies demonstrated that, with increasing substrate size, the energy of 2EN and substrate co-binding in the active site became unfavorable precisely at the point where 2EN became a competitive inhibitor. Hierarchical docking revealed potential allosteric inhibition sites separate from the substrate binding site.

Mapping of cytochrome P450 2B4 substrate binding sites by photolabile probe 3-azidiamantane: identification of putative substrate access regions.

To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu359-Lys373), peptide II (Leu30-Arg48), peptide III (Phe127-Arg140), and peptide IV (Arg434-Arg443). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg133, marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.

Structural insight into the altered substrate specificity of human cytochrome P450 2A6 mutants.

Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B'-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.