Date of Award

5-2020

Document Type

Thesis

Degree Name

Bachelor of Science

College/School

College of Science and Mathematics

Department/Program

Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

Jaclyn Catalano

Committee Member

Yvonne Gindt

Committee Member

Eli Lee

Abstract

Cytochrome P450 is an enzyme mainly found in liver cells. It plays a key role in the breakdown of drugs and toxins by hydroxylating substrates making the substrates more water soluble. P450s are also involved in cholesterol and hormone synthesis. The third P450 from bacterium Bacillus Megaterium (BM3) is used as a model P450 enzyme due to its structural similarity to human P450s and due to its efficiency in the hydroxylation of fatty acids. Within the enzyme’s active site, substrates can occupy a distal or proximal conformation monitored by UV-vis spectroscopy. The proximal conformation is more effective when it comes to hydroxylation so a double mutant (A82F/F87A) was developed and it was hypothesized that the mutation would allow for the proximal conformation to be more prevalent and increase the catalytic efficiency. Enzymatic Studies with palmitic (C16:0), eicosanoic (C20:0), and arachidonic (C20:4) acids as substrates were performed for the wild-type and mutant enzyme. Michaelis–Menten Kinetics were used to determine Km, kcat, and catalytic efficiency of the enzymes. The results of the experiments have not supported the hypothesis and the wild-type enzyme has shown to be a more efficient enzyme than the mutant. However, an overall increase in the catalytic efficiency of both mutant and wild type was noted with increasing temperature. Future studies include further experimentation with different chain length and saturation levels of fatty acids as well as looking at the products of enzymatic reactions as a function of temperature by LC/MS

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