Decreased substrate affinity upon alteration of the substrate‐docking region in cytochrome P450BM‐3

A mutation at the surface of the substrate access channel which dramatically decreases the affinity for some fatty acids in P450(BM-3) was discovered by random mutagenesis. The mutation introduced, proline-25 to glutamine, is in close proximity to the arginine-47 residue thought to be responsible for the initial docking of fatty acid substrates. The P25Q mutant displays an affinity for palmitate which is approximately 100-fold weaker than the wild-type enzyme. In addition to its altered substrate affinity, P25Q also exhibits altered hydroxylation specificity and carbon monoxide recombination kinetics in the substrate-free form.