The long-term goal of this project is to elucidate the interrelationships between the structure and conformational dynamics of cytochrome P450 enzymes, their catalytic mechanisms, and the consequences of these interactions in terms of catalytic outcome and substrate specificity. The phase of the work proposed here has three major goals: (a) To utilize NMR and fluorescent probes to elucidate the conformational range of bacterial P450 enzymes and the nature of the environment in the vicinity of the thiolate-iron ligand;(b) to investigate the mechanisms by which mutations distant from the active site alter the catalytic process, focusing on the possible role of coupled protein motions and conformational links;(c) to further stabilize the monomeric state of solubilized human P450 enzymes and to extend the NMR and fluorescent conformational probe studies to these proteins;and (d) to apply cutting edge computational docking techniques to the conformational ensembles emerging from the combined use of molecular dynamics and NMR to construct refined protocols for the prediction and rationalization of cytochrome P450 substrate and inhibitor specificity, focusing initially on CYP2C9 The work proposed in this application rests on a number of technical advances. First, efforts to determine the structures of membrane-bound human P450 enzymes have succeeded in the past decade and more than a dozen P450 enzyme structures in various ligation states are now available. Second, the rapid progress in NMR techniques has allowed us to demonstrate the utility of site-specific NMR probes in conformational analysis of proteins as large as the P450 enzymes. Third, methods for the site-specific incorporation of unnatural and labeled amino acids now make it possible to tag individual positions in a protein with NMR or fluorescent probes. Fourth, advances in computational algorithms and the increase in computer speed now allow long-term molecular dynamics studies and computational docking of flexible ligands in flexible active sites. This is a broad study of the interplay of conformational dynamics and its role in modulating catalysis and determining substrate specificity in a set of enzymes that are notoriously malleable and challenging. The results should have a major impact in the pharmaceutical industry, for which the P450 specificity for a given drug candidate can determine whether it progresses or is discarded. They will also impact the design of inhibitors for P450 enzyme targeted in cancer and other diseases, and the design of P450 catalysts for biotechnological applications. Beyond P450, the conformational issues dealt with in this project and the techniques being explored have broader applicability and will have an impact in completely different areas of biochemistry and biology.
Cytochrome P450 enzymes fulfill critical roles in the metabolism of drugs and environmental chemicals, the biosynthesis of endogenous factors such as the sterol hormones, and the termination of the action of endogenous factors such as retinoic acid. They are of major practical interest because: (a) they are important targets for clinically used antifungal, antiparasitic, and anticancer drugs, (b) they are major determinants of drug pharmacokinetics and drug action, (c) they are responsible for a broad range of metabolism-dependent drug toxicities, and (d) they have high potential as biotechnological catalysts. The ability to predict P450 substrate and inhibitor specificity, and the effect of mutations on catalytic function, is central to all of these areas.
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