Investigating the Effects and Mechanisms of Interactions between Cytochrome P450 2B4, Cytochrome P450 2E1 and Cytochrome P450 Reductase

Abstract

A requirement for cytochrome P450 (CYP or P450)-mediated drug metabolism is the association of P450s with cytochrome P450 reductase (CPR). Although P450s form a 1:1 complex with CPR, they exist in excess over CPR in the endoplasmic reticulum (ER). Very little is known about the effect of less than stoichiometric amounts of CPR relative to P450 in the ER on the interaction of P450s with CPR and substrate metabolism. Equally little is known about the mechanism of interaction of P450s with CPR since much of our knowledge regarding the specific residues that mediate this interaction stems from a limited number of mutagenesis studies. In this thesis we developed methodology to directly probe the CYP2B4-CPR binding interface and demonstrated novel roles for residues V267 and L270 of CYP2B4 in binding CPR. We harnessed this knowledge to engineer a CYP2B4 with greater rates of reduction and substrate metabolism. We also found that CYP2E1, an inducible P450 isoform, significantly inhibited the catalytic activity of CYP2B4 in a concentration-dependent manner. We proposed a preliminary model to explain the inhibitory behavior of CYP2E1 toward CYP2B4 that was based on two key findings: 1) direct CYP2B4-CYP2E1 interactions alone do not lead to inhibition of CYP2B4 activity in the presence of saturating concentrations of substrate and 2) CYP2E1 has a higher affinity for CPR in the presence of CYP2B4. In this model we suggested that CYP2E1 and CYP2B4 associate to form a CYP2B4-CYP2E1 complex that interacts with the functional site of CPR with a higher affinity than CYP2E1 alone, and this complex may allow CYP2E1 to compete with CYP2B4 for CPR. Taken together, the work presented in this thesis establishes a new approach to the identification of amino acid residues that mediate redox-partner recognition and demonstrates how these residues can be used to enhance P450 activity. Additionally, these reports provide us with valuable insights into the potential for protein-protein interactions in the P450 system to confound in vitro – in vivo drug metabolism extrapolations and may play an important role in improving our ability to predict in vivo drug clearance and drug-drug interactions from in vitro data.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/94027/1/ckenaan_1.pd