NMR Studies of Ligand Binding to P450eryF Provides Insight into the Mechanism of Cooperativity
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 íM, 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 Hillequation- 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 lowaffinity 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.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 883213
- Report Number(s):
- PNNL-SA-48932; 8000; KP1303000
- Journal Information:
- Biochemistry, 45(6):1673-1684, Journal Name: Biochemistry, 45(6):1673-1684
- Country of Publication:
- United States
- Language:
- English
Similar Records
Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography
Biostructural chemistry of magnesium ion: Characterization of the weak binding sites on tRNA sup Phe (yeast). Implications for conformational change and activity