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Structural evidence: a single charged residue affects substrate binding in cytochrome P450 BM-3
Jaclyn Catalano1, Kianoush Sadre-Bazzaz, Gabriele A Amodeo
1Department of Chemistry, Columbia University , 3000 Broadway, New York, New York 10027, United States.
Biochemistry
|July 9, 2013
Summary
Researchers studied cytochrome P450 BM-3 enzyme dynamics and substrate binding. A key arginine residue (R47) was found crucial for binding N-palmitoylglycine (NPG), impacting enzyme activity.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Cytochrome P450 BM-3 is a bacterial enzyme similar to mammalian P450s, known for efficient fatty acid hydroxylation.
- Understanding enzyme-substrate interactions and dynamics is critical, as P450 crystal structures often depict inactive states.
Purpose of the Study:
- To investigate the role of arginine 47 (R47), the sole charged residue in the P450 BM-3 active site, in substrate binding and catalysis.
- To elucidate the structural basis of enzyme-substrate complex formation using N-palmitoylglycine (NPG).
Main Methods:
- Determined a novel crystal structure of cytochrome P450 BM-3 complexed with NPG.
- Performed site-directed mutagenesis of R47 to glutamine (R47Q), glutamic acid (R47E), and lysine (R47K).
- Conducted crystallographic studies and enzymatic activity assays on the wild-type and mutant enzymes.
Main Results:
- A crystal structure revealed an unexpected bidentate ion pair between NPG and R47.
- The R47E mutation significantly reduced enzyme activity and binding affinity, with the glutamic acid residue obstructing the active site entrance.
- Mutations R47Q and R47K showed minor changes in catalytic rate (kcat) but substantial increases in Michaelis constant (Km) and dissociation constant (Kd).
Conclusions:
- Arginine 47 (R47) plays a significant role primarily in the binding of NPG to P450 BM-3.
- The determined crystal structure (4KPA) likely represents an early stage in the P450 catalytic cycle, highlighting initial substrate binding.
- The charge and positioning of R47 are critical for efficient substrate recognition and potentially catalysis.
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