Determining the Role of E267: Proton Transfer or Substrate Selectivity
Presentation Type
Poster
Faculty Advisor
Jaclyn Catalano
Access Type
Event
Start Date
26-4-2024 12:45 PM
End Date
26-4-2024 1:44 PM
Description
Cytochrome P450 is a heme protein essential in the metabolization of drugs. CYPs are being investigated as drug targets for anticancer prodrugs because they have the ability to convert the drug into its active form in the body. A bacterial model of cytochrome P450 is being used to mimic the active site of CYP2S1, which is overexpressed in breast cancer cells. First, we studied the role of E267 in Cytochrome P450 BM-3 by creating single mutations at residue 267. We hypothesized that mutations to E267 will alter the kinetics of Cytochrome P450 by interfering with proton transfer, binding, or activity. 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. Second, we determined the enzymatic parameters for the E267V mutation with substrate N-palmitoylglycine (NPG). The mutation had higher catalytic efficiency values than the wild type protein. This confirms that the mutations will alter the kinetics which could be due to the active site of the protein becoming wider. We then ran kinetic assays with another substrate noscapine, which is a bulkier substrate. We found that three of our mutations had higher activity than the wild type protein. Overall, it was seen that E267 is not involved in the mechanism and supports the hypothesis that the residue is involved in widening the active site.
Determining the Role of E267: Proton Transfer or Substrate Selectivity
Cytochrome P450 is a heme protein essential in the metabolization of drugs. CYPs are being investigated as drug targets for anticancer prodrugs because they have the ability to convert the drug into its active form in the body. A bacterial model of cytochrome P450 is being used to mimic the active site of CYP2S1, which is overexpressed in breast cancer cells. First, we studied the role of E267 in Cytochrome P450 BM-3 by creating single mutations at residue 267. We hypothesized that mutations to E267 will alter the kinetics of Cytochrome P450 by interfering with proton transfer, binding, or activity. 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. Second, we determined the enzymatic parameters for the E267V mutation with substrate N-palmitoylglycine (NPG). The mutation had higher catalytic efficiency values than the wild type protein. This confirms that the mutations will alter the kinetics which could be due to the active site of the protein becoming wider. We then ran kinetic assays with another substrate noscapine, which is a bulkier substrate. We found that three of our mutations had higher activity than the wild type protein. Overall, it was seen that E267 is not involved in the mechanism and supports the hypothesis that the residue is involved in widening the active site.