A survey of active site access channels in cytochromes P450

In cytochrome P450s, the active site is situated deep inside the protein next to the heme cofactor, and is often completely isolated from the surrounding solvent. To identify routes by which substrates may enter into and products exit from the active site, random expulsion molecular dynamics simulations were performed for three cytochrome P450s: CYP101, CYP102A1 and CYP107A1 [J. Mol. Biol. 303 (2000) 797; Proc. Natl. Acad. Sci. USA 99 (2002) 5361]. Amongst the different pathways identified, one pathway was found to be common to all three cytochrome P450s although the mechanism of ligand passage along it was different in each case and apparently adapted to the substrate specificity of the enzyme. Recently, a number of new crystal structures of cytochrome P450s have been solved. Here, we analyse the open channels leading to the active site that these structures reveal. We find that in addition to showing the common pathway, they provide experimental evidence for the existence of three additional channels that were identified by simulation. We also discuss how the location of xenon binding sites in CYP101 suggests a role for one of the pathways identified by molecular dynamics simulations as a route for gaseous species, such as oxygen, to access the active site.     

Do mammalian cytochrome P450s show multiple ligand access pathways and ligand channelling?

Understanding substrate binding and product release in cytochrome P450 (CYP) enzymes is important for explaining their key role in drug metabolism, toxicity, xenobiotic degradation and biosynthesis. Here, molecular simulations of substrate and product exit from the buried active site of a mammalian P450, the microsomal CYP2C5, identified a dominant exit channel, termed pathway (pw) 2c. Previous simulations with soluble bacterial P450s showed a different dominant egress channel, pw2a. Combining these, we propose two mechanisms in CYP2C5: (i) a one-way route by which lipophilic substrates access the enzyme from the membrane by pw2a and hydroxylated products egress along pw2c; and (ii) a two-way route for access and egress, along pw2c, for soluble compounds. The proposed differences in substrate access and product egress routes between membrane-bound mammalian P450s and soluble bacterial P450s highlight the adaptability of the P450 fold to the requirements of differing cellular locations and substrate specificity profiles.     

Multiple molecular recognition mechanisms. Cytochrome P450--a case study

Biomolecular recognition is complex. The balance between the different molecular properties that contribute to molecular recognition, such as shape, electrostatics, dynamics and entropy, varies from case to case. This, along with the extent of experimental characterization, influences the choice of appropriate computational approaches to study biomolecular interactions. Here, we present computational studies of cytochrome P450 enzymes and their interactions with small molecules and with other proteins. These interactions exemplify some of the diversity of molecular determinants of binding affinity and specificity observed for proteins and we discuss some of the challenges that they pose for molecular modelling and simulation.