The chemistry catalyzed by cytochromes P450 (activation of molecular oxygen to react with organic molecules) is inherently hazardous to the organism, and must be tightly regulated or take place in a controlled environment. Structural and dynamic changes that take place in the course of the catalytic cycle of the P450 enzyme are part of this regulation, and help determine enzyme specificity and efficiency. While crystal structures have been determined for many P450s, and provide an important starting point for understanding structure-function relationships, methodology is lacking for monitoring changes in structure and dynamics as a function of substrate and effector binding or for rapid characterization of active sites of P450s and their interactions with substrates and inhibitors. We apply multidimensional nuclear magnetic resonance (NMR) and tandem mass spectrometry (MS-MS) to P450 enzymes to fill this need. Extensive sequential 1H, 15N and 13C resonance assignments have been made in cytochrome P450cam (CYP101). A discrete conformational change occurs upon binding of effector to CYP101 that reorients the substrate appropriately for chemistry, and details of this conformational change have been elucidated by a combination of NMR, mutagenesis and simulations. MS-MS has been used to localize redox-dependent changes in local protein dynamics in CYP101 by hydrogen/deuterium exchange. During the next project period, NMR assignments of CYP101 will be extended to regions of slow amide exchange and paramagnetic broadening. MS-MS and NMR will be used to identify dynamic "hot-spots" important for substrate binding. The structure of effector-bound CYP101 will be determined using residual dipolar couplings. NMR resonances in the active sites of P450s can be easily distinguished by comparison of paramagnetic and diamagnetic forms. The binding of substrates and inhibitors in the active sites of mammalian P450 enzymes with CYP2B4 and CYP3A4 will be characterized. Methodology developed during the current period for NMR structural characterization of paramagnetic metalloproteins will be further refined.

Public Health Relevance

Project Narrative Cytochrome P450 monooxygenases are critical in many human physiological processes, including drug metabolism and activation, steroid and arachadonic acid biosynthesis, and "toxic waste disposal", that is, degradation of foreign compounds prior to excretion. Understanding the activity of these enzymes is important for predicting the behavior of inhibitors and substrates as a part of drug design. This project is aimed at understanding these enzymes to aid in the design of more effective pharmaceuticals.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM044191-21
Application #
8272544
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Wehrle, Janna P
Project Start
1990-04-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2014-05-31
Support Year
21
Fiscal Year
2012
Total Cost
$309,712
Indirect Cost
$113,692
Name
Brandeis University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454
Pochapsky, Thomas C (2014) Examining how enzymes self-organize in a membrane. Proc Natl Acad Sci U S A 111:3659-60
Friedman, Erin J; Wang, Helen X; Jiang, Kun et al. (2011) Acireductone dioxygenase 1 (ARD1) is an effector of the heterotrimeric G protein beta subunit in Arabidopsis. J Biol Chem 286:30107-18
Asciutto, Eliana K; Dang, Marina; Pochapsky, Susan Sondej et al. (2011) Experimentally restrained molecular dynamics simulations for characterizing the open states of cytochrome P450cam. Biochemistry 50:1664-71
Dang, Marina; Pochapsky, Susan Sondej; Pochapsky, Thomas C (2011) Spring-loading the active site of cytochrome P450cam. Metallomics 3:339-43
Pochapsky, Thomas C; Kazanis, Sophia; Dang, Marina (2010) Conformational plasticity and structure/function relationships in cytochromes P450. Antioxid Redox Signal 13:1273-96
Asciutto, Eliana K; Madura, Jeffry D; Pochapsky, Susan Sondej et al. (2009) Structural and dynamic implications of an effector-induced backbone amide cis-trans isomerization in cytochrome P450cam. J Mol Biol 388:801-14
Pochapsky, Susan Sondej; Dang, Marina; OuYang, Bo et al. (2009) Redox-dependent dynamics in cytochrome P450cam. Biochemistry 48:4254-61
Hamuro, Yoshitomo; Molnar, Kathleen S; Coales, Stephen J et al. (2008) Hydrogen-deuterium exchange mass spectrometry for investigation of backbone dynamics of oxidized and reduced cytochrome P450cam. J Inorg Biochem 102:364-70
OuYang, Bo; Pochapsky, Susan Sondej; Dang, Marina et al. (2008) A functional proline switch in cytochrome P450cam. Structure 16:916-23
Pochapsky, Susan Sondej; Sunshine, Joel C; Pochapsky, Thomas C (2008) Completing the circuit: direct-observe 13C,15N double-quantum spectroscopy permits sequential resonance assignments near a paramagnetic center in acireductone dioxygenase. J Am Chem Soc 130:2156-7

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