Drug Interactions of Human and Bacterial Cytochrome P450?s Probed by Pulsed Electron Paramagnetic Spectroscopy
EMSL Project ID
8000
Abstract
We are interested in using pulsed electron paramagnetic resonance (EPR) techniques that are available at the EMSL facility to probe drug interactions to and structure of cytochrome P450?s. We are interested in looking at a wide range of cytochrome P450?s that are involved in drug metabolism. Below are a couple of studies that we would like to perform, using pulsed EPR techniques and their potential outcome.Human cytochrome P450 2C9 (CYP2C9) is known to metabolize a wide range of drugs to their non-toxic form, including ibuprofen and warfarin. We have a mutant of this enzyme that produces absorbance and continuous-wave EPR spectra that is consistent with an intramolecular covalent linkage between a histidine of CYP2C9 and the Fe3+ of the heme in CYP2C9. There are pulsed EPR techniques (i.e. hyperfine sublevel correlation (HYSCORE) spectroscopy, electron nuclear double resonance (ENDOR), and electron nuclear quadruple resonance (ENQOR)) that we would like to use at the EMSL facility that can conclusively show that this linkage is indeed between a histidine and Fe3+ (e.g. Rao, B.K., et al., Biochemistry, 2000. 39(12): p. 3285-3296). The results of these experiments will provide us with greater insight into the structure of the active site.
Human cytochrome P450 3A4 (CYP3A4) and its bacterial analog, cytochrome P450 eryF (CYPeryF) metabolize >60% of known hydrophobic drugs and hormones. Of these compounds, there are many that are known to form a covalent nitrogen linkage to the Fe3+ of the heme. We would like to use pulsed EPR techniques to probe the coupling between the nitrogen of the drug, 9-aminophenanthrene, and the unpaired electron of the Fe3+ heme in CYPeryF and CYP3A4. This should provide us with insight into the binding and orientation of this drug in the active sites of these cytochrome P450?s. Since an x-ray crystal structure of CYPeryF with 9-aminophenanthrene is available, a comparison of binding of 9-aminophenanthrene with CYPeryF and CYP3A4 will be particularly useful for getting a better understanding of the similarities and differences between their active sites.
Project Details
Project type
Capability Research
Start Date
2004-03-15
End Date
2006-04-10
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members