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Cytochrome P450-ligand interactions

Objective

Cytochrome P450 enzymes are hemoprotein monooxygenases that play a critical role in the synthesis and degradation of many physiologically important compounds and xenobiotics. They thus have considerable potential in the biotechnology industry where they may be of value for the large scale synthesis of drugs and other chemicals or as catalysts for the degradation of environmental pollutants. Essential for the exploitation of cytochrome P450 for such purposes is a thorough knowledge of the structural, energetic, dynamic and functional properties of the enzyme and its interactions with ligands such as substrates and inhibitors. The aim of this project is to provide a detailed understanding of these properties of cytochrome P450cam (P450). The research should thus provide a firm foundation for the exploitation of P450s as novel catalysts. The project is also designed to provide insights into the nature of protein-ligand interactions in general and to result in the development of new methods to study and design protein ligands.
Three main topics are under study in the project:
(i) Characterization of the energy landscape of P450 and the transition to the inactive P420 form. The P450-P420 transition was shown to involve an a-helix to b-sheet transition and result in significant changes in the active site structure and a P420 structure dependent on the means of inducing its formation.
(ii) Elucidation of the energetic and structural roles played by water molecules and ions in determining the protein structure and the binding and catalysis of ligands. Molecular dynamics simulation methods were used to compute free energies of hydrating interfacial cavities in the active site and the hydration occupancy of the substrate-free active site. The latter is consistent with the crystallographic assignment to model a disordered water cluster.
Coupling between the buried active site and the surface cation binding site (with weak preference for K+) was observed with mutual stabilization on binding of the natural substrate, 1R-camphor, and K+ ions respectively.
(iii) Elucidation of the mechanism of access, binding and catalysis of substrates in the active site of P450, and the design of ligands to bind to P450 and to be used as tools for probing the properties of P450. The importance of saltbridges mediated by D251 in controlling substrate access to the active site was shown in spectroscopic and photoacoustic measurements. Electrostatic calculations show these salt-links to be exceptionally stable.
Binding of a series of camphor analogues was characterized and the structure-based design of a substrate analogue was tested. A designed compound with higher affinity than 1R-camphor was obtained and found to bind in a similar position, displacing about as much water from the active site. It is a substrate which although with lower activity than camphor, demonstrates little uncoupling and surprisingly has a different mechanism from camphor.
The structures of ternary complexes of P450, camphor and oxygen were solved and give evidence for the existence of an oxyferryl intermediate.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Address
Meyerhofstrasse 1
Heidelberg
Germany

Participants (5)

Centre National de la Recherche Scientifique (CNRS)
France
Address
263 Avenue Du Général Leclerc
35042 Rennes
Institut National de la Santé et de la Recherche Médicale (INSERM)
France
Address
13 Rue Pierre Et Marie Curie
75005 Paris
MAX DELBRUCK CENTER FOR MOLECULAR MEDICINE
Germany
Address
Robert-roessle-strasse 10
13092 Berlin
MAX-PLANCK-GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN E.V.
Germany
Address
Jahnstraße 29
69120 Heidelberg
THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
United Kingdom
Address
Mansfield Road
Oxford