Role of E267 in Proton Transfer
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 primarily found in the liver, responsible for metabolizing drugs in the body. Among its various isoforms, CYP2S1 stands out as a mammalian cytochrome P450 (CYP) notably overexpressed in cancer cells, presenting a potential drug target. Unlike other CYPs, CYP2S1 exhibits distinct chemistry and mechanisms, which we hypothesize may be attributed to the absence of a conserved acidic residue. To investigate the role of this conserved acidic residue, we've opted to employ a bacterial model (BM-3) since its easier to express and purify. BM-3’s native enzyme is glutamic acid (E) at residue 267. Through site-directed mutagenesis, we generated single mutations at residue 267, namely E267K, E267V, and E267D. Our primary research question revolves around determining whether E267 is involved in proton transfer. Initial deuterium studies have indicated a decrease in catalytic activity among the mutations, with the exception of E267V. Consequently, we conducted reproducibility studies using deuterium oxide with E267V to ascertain if it would yield consistent results, particularly in exhibiting enhanced kinetics in deuterium oxide. These experiments aim to shed light on the specific contributions of amino acid substitutions to the mechanism of E267 in proton transfer.
Role of E267 in Proton Transfer
Cytochrome P450 is a heme protein primarily found in the liver, responsible for metabolizing drugs in the body. Among its various isoforms, CYP2S1 stands out as a mammalian cytochrome P450 (CYP) notably overexpressed in cancer cells, presenting a potential drug target. Unlike other CYPs, CYP2S1 exhibits distinct chemistry and mechanisms, which we hypothesize may be attributed to the absence of a conserved acidic residue. To investigate the role of this conserved acidic residue, we've opted to employ a bacterial model (BM-3) since its easier to express and purify. BM-3’s native enzyme is glutamic acid (E) at residue 267. Through site-directed mutagenesis, we generated single mutations at residue 267, namely E267K, E267V, and E267D. Our primary research question revolves around determining whether E267 is involved in proton transfer. Initial deuterium studies have indicated a decrease in catalytic activity among the mutations, with the exception of E267V. Consequently, we conducted reproducibility studies using deuterium oxide with E267V to ascertain if it would yield consistent results, particularly in exhibiting enhanced kinetics in deuterium oxide. These experiments aim to shed light on the specific contributions of amino acid substitutions to the mechanism of E267 in proton transfer.