Metalloenzyme Mechanisms

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DESCRIPTION (provided by applicant): Our major research goal is elucidating the mechanisms of molecular recognition in metalloprotein systems, including the recognition of proteins with other proteins, with small molecule substrates/inhibitors and with the lipid membrane components that establish and control the assembly of supra-molecular membrane complexes that affect all aspects of recognition and catalysis. Efforts focus on the human hepatic cytochromes P450 that are central players in drug metabolism. Due to their important role in human health and disease, these enzyme systems have occupied investigators for many decades. Despite an intense research effort, a complete molecular understanding of the biophysical and biochemical mechanisms of P450 catalysis remain important goals. One obstacle has been that the human cytochrome P450s are integral membrane proteins. While much has been learned by investigating their function in native membrane preparations or by detergent solubilization, critical knowledge on the role of the membrane in facilitating function and the basis for substrate recognition is lacking. In a novel approach, we surmount this challenge by making make use of the Nanodisc system (developed under support from this grant) that self-assembles the membrane protein target into a discoidal nanometer size lipid bilayer that is soluble in aqueous solution. We use a variety of biophysical approaches in order to define the mechanism of protein self-assembly into these robust bilayer structures and to reveal the topology of P450 and its redox partner, cytochrome P450 reductase (CPR). In seeking to understand the details of how lipids interact with proteins and multi-protein complexes, we define the mechanisms of complex formation between human hepatic P450 and CPR as well as explore the potential role of P450 oligomerization in drug metabolism. The human P450 detoxifying enzymes require a highly plastic active site in order to functionally accommodate a wide spectrum of substrate structures. The ability to bind and metabolize multiple substrates in the P450 active site is the origin of "drug-drug" interactions, which in many cases, is responsible for deleterious side effects and limitations in the therapeutic window between efficacy and toxicity. Hence, understanding this aspect of molecular recognition in the precise mechanisms and magnitude of cooperative substrate binding is also an important goal of our future work. These efforts to reveal the principles of molecular recognition in metalloprotein mechanisms are linked through application of a breadth of biophysical, biochemical and structural techniques to reveal the molecular details of self-assembly, the role of the membrane environment and protein conformational determinants of cytochrome P450 catalysis.