Importance of a Conserved Acidic Residue in Cytochrome P450
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
Cytochromes P450 (CYP) are enzymes that are responsible for metabolism of many different compounds, including drugs and toxins, and involved in the synthesis of steroids and vitamins. CYP2S1, a particular variant of CYP, has been shown to be overexpressed in certain cancer cells and has been investigated as a prodrug target (activated by CYPs only in cancer cells). CYP2S1, unlike most known CYPs, has been shown to catalyze the reduction of substrates instead of oxidation. We hypothesize that this unique chemistry is due to the lack of the acidic residue before the conserved threonine, therefore we replaced the acidic residue (glutamic acid) with a nonpolar residue (valine) to see if the activity would be altered. Enzymatic Reactions with NADPH (cofactor-electron source), substrate (NPG) and enzyme (at different concentrations) were carried out. Experiments were done at pH=6.5, 7.5, and 8.5. We observed that the rate of the reaction increases with increasing enzyme concentration and our results agree with previous studies on relative activity pH profiles performed in the lab which shows a different pH profile for the wild type enzyme compared to the valine mutant. The enzyme had similar activity with the valine mutant and wildtype enzyme showing the acidic residue, glutamic acid, is not essential for activity. However, the difference in pH profile shows that the acidic amino acid may have a different role potentially in substrate binding and/or size of the active site binding pocket.
Importance of a Conserved Acidic Residue in Cytochrome P450
Cytochromes P450 (CYP) are enzymes that are responsible for metabolism of many different compounds, including drugs and toxins, and involved in the synthesis of steroids and vitamins. CYP2S1, a particular variant of CYP, has been shown to be overexpressed in certain cancer cells and has been investigated as a prodrug target (activated by CYPs only in cancer cells). CYP2S1, unlike most known CYPs, has been shown to catalyze the reduction of substrates instead of oxidation. We hypothesize that this unique chemistry is due to the lack of the acidic residue before the conserved threonine, therefore we replaced the acidic residue (glutamic acid) with a nonpolar residue (valine) to see if the activity would be altered. Enzymatic Reactions with NADPH (cofactor-electron source), substrate (NPG) and enzyme (at different concentrations) were carried out. Experiments were done at pH=6.5, 7.5, and 8.5. We observed that the rate of the reaction increases with increasing enzyme concentration and our results agree with previous studies on relative activity pH profiles performed in the lab which shows a different pH profile for the wild type enzyme compared to the valine mutant. The enzyme had similar activity with the valine mutant and wildtype enzyme showing the acidic residue, glutamic acid, is not essential for activity. However, the difference in pH profile shows that the acidic amino acid may have a different role potentially in substrate binding and/or size of the active site binding pocket.