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Title: NMR Studies of Ligand Binding to P450 eryF Provides Insight into the Mechanism of Cooperativity

Abstract

Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450 eryF. UV/vis absorbance spectra of P450 eryF demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of KS = 4 and 200 μM, respectively. The broadening and shifting observed in the 2D-{ 1H, 15N}-HSQC-monitored titrations of 15N-Phe-labeled P450 eryF with 9-AP and TST indicated binding on intermediate and fast chemical exhange time scales, respectively, which was consistent with the Hill-equation-derived KS values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands ''contact'' the same phenylalanines within the active site of P450 eryF. This findingmore » is in agreement with X-ray crystal structures of bound P450 eryF showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the low-affinity TST molecule or multiple TST-bound P450 eryF conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [1];  [2];  [1];  [1]
  1. Univ. of Washington, Seattle, WA (United States)
  2. Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
Publication Date:
Research Org.:
Environmental Molecular Sciences Laboratory (EMSL), Richland, WA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1152355
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 45; Journal Issue: 6
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Roberts, Arthur G., Diaz, M. Delores, Lampe, Jed N., Shireman, Laura M., Grinstead, Jeffrey S., Dabrowski, Michael J., Pearson, Josh T., Bowman, Michael K., Atkins, William M., and Campbell, A. Patricia. NMR Studies of Ligand Binding to P450eryF Provides Insight into the Mechanism of Cooperativity. United States: N. p., 2006. Web. doi:10.1021/bi0518895.
Roberts, Arthur G., Diaz, M. Delores, Lampe, Jed N., Shireman, Laura M., Grinstead, Jeffrey S., Dabrowski, Michael J., Pearson, Josh T., Bowman, Michael K., Atkins, William M., & Campbell, A. Patricia. NMR Studies of Ligand Binding to P450eryF Provides Insight into the Mechanism of Cooperativity. United States. doi:10.1021/bi0518895.
Roberts, Arthur G., Diaz, M. Delores, Lampe, Jed N., Shireman, Laura M., Grinstead, Jeffrey S., Dabrowski, Michael J., Pearson, Josh T., Bowman, Michael K., Atkins, William M., and Campbell, A. Patricia. Wed . "NMR Studies of Ligand Binding to P450eryF Provides Insight into the Mechanism of Cooperativity". United States. doi:10.1021/bi0518895.
@article{osti_1152355,
title = {NMR Studies of Ligand Binding to P450eryF Provides Insight into the Mechanism of Cooperativity},
author = {Roberts, Arthur G. and Diaz, M. Delores and Lampe, Jed N. and Shireman, Laura M. and Grinstead, Jeffrey S. and Dabrowski, Michael J. and Pearson, Josh T. and Bowman, Michael K. and Atkins, William M. and Campbell, A. Patricia},
abstractNote = {Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450eryF. UV/vis absorbance spectra of P450eryF demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of KS = 4 and 200 μM, respectively. The broadening and shifting observed in the 2D-{1H,15N}-HSQC-monitored titrations of 15N-Phe-labeled P450eryF with 9-AP and TST indicated binding on intermediate and fast chemical exhange time scales, respectively, which was consistent with the Hill-equation-derived KS values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands ''contact'' the same phenylalanines within the active site of P450eryF. This finding is in agreement with X-ray crystal structures of bound P450eryF showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the low-affinity TST molecule or multiple TST-bound P450eryF conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.},
doi = {10.1021/bi0518895},
journal = {Biochemistry},
number = 6,
volume = 45,
place = {United States},
year = {Wed Feb 01 00:00:00 EST 2006},
month = {Wed Feb 01 00:00:00 EST 2006}
}