Determine the role of an acidic residue in Cytochrome P450

Cytochrome P450 is a heme protein essential in the metabolization of drugs. CYPs have been investigated as drug targets for anticancer prodrugs (to be activated by P450 only in cancer cells). We are using a bacterial model of cytochrome P450 to mimic the active site of CYP2S1, which is overexpressed in breast cancer cells, by creating single mutations at residue 267. First, we studied the role of E267 in Cytochrome P450 BM-3. We hypothesize that mutations to E267 will alter the kinetics of Cytochrome P450 by interfering with proton transfer in the mechanism and/or be involved in expanding the binding pocket to incorporate bulkier substrates. Acidic residue E267 was mutated to valine, methionine, aspartic acid, and lysine. Michaelis Menten kinetics and the effect on the kinetics as a function of pH was studied. The findings show that all three mutants have a similar pH profile compared to the wild-type and there is no activity for the methionine mutant. Second, we determined the enzymatic parameters for one mutation with substrate N-palmitoylglycine (NPG). The mutation E267V had a higher Km, kcat, and catalytic efficiency values than the wild type protein. This confirms that the mutations will alter the kinetics and could be due to the active site of the protein becoming wider. We then ran kinetic assays with another substrate noscapine, which is also an anti cancer prodrug. We found that three of our mutations had higher activity than the wild type protein.