Role of Enzyme Flexibility in Ligand Access and Egress to Active Site: Bias-Exchange Metadynamics Study of 1,3,7-Trimethyluric Acid in Cytochrome P450 3A4

Although the majority of enzymes
have buried active sites, very
little is known about the energetics and mechanisms associated with
substrate and product channeling in and out. Gaining direct information
about these processes is a challenging task both for experimental
and theoretical techniques. Here, we present a methodology that enables
following of a ligand during its passage to the active site of cytochrome
P450 (CYP) 3A4 and mapping of the free energy associated with this
process. The technique is based on a combination of a bioinformatics
tool for identifying access channels and bias-exchange metadynamics
and provides converged free energies in good agreement with experimental
data. In addition, it identifies the energetically preferred escape
routes, limiting steps, and amino acids residues lining the channel.
The approach was applied to mapping of a complex channel network in
a complex environment, i.e., CYP3A4 attached to a lipid bilayer mimicking
an endoplasmic reticulum membrane. The results provided direct information
about the energetics and conformational changes associated with the
ligand channeling. The methodology can easily be adapted to study
channeling through other flexible biomacromolecular channels.