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Structural evidence for a functionally relevant second camphor binding site in P450cam: model for substrate entry
Huili Yao1, Christopher R McCullough, Aurora D Costache
1Department of Chemistry, Marquette University, Milwaukee, Wisconsin 53201, USA.
Proteins
|June 29, 2007
Summary
Cytochrome P450cam may use a two-step substrate binding process. This mechanism minimizes heme exposure by allowing transient binding near the active site before full entry, consistent with a two-site model.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Cytochrome P450cam serves as a model for the P450 gene family.
- Understanding substrate entry into the active site is crucial for P450 function.
- Minimizing reactive heme exposure during substrate binding is a key challenge.
Purpose of the Study:
- To investigate the substrate binding mechanism of P450cam.
- To test the hypothesis of a two-step binding process involving an initial transient site.
- To elucidate how P450cam minimizes heme exposure during substrate access.
Main Methods:
- Computational docking of a second camphor molecule into the P450cam crystal structure.
- Nuclear Magnetic Resonance (NMR) T1 relaxation measurements.
- [(1)H-(13)C] HSQC titrations of labeled P450cam.
- NMR chemical shift probing with a heme-blocked P450cam-pyridine complex.
Main Results:
- A potential substrate entry pocket was identified on the F-G helix side, 16 Å from the heme.
- NMR data confirmed the presence of a second camphor binding site distinct from the active site.
- Camphor binding outside the active site was confirmed with a heme-blocked enzyme, yielding a Kd of 43 μM.
Conclusions:
- P450cam likely employs a two-site binding mechanism for substrate entry.
- This mechanism allows the enzyme to remain in a closed conformation longer, minimizing heme exposure.
- The proposed model is analogous to mechanisms observed in other P450 enzymes like CYP3A4.
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