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Date of Award

Spring 2023

Document Type

Masters Thesis

Department or Program Affiliation

Chemistry

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Spiegel, P. Clint

Second Advisor

McCarty, Jay

Third Advisor

Antos, John M.

Abstract

Cytosine Deaminases (CD) are a class of enzymes found in prokaryotes and fungi that have been studied in the past due to their ability to deaminate the prodrug 5-fluorocytosine (5-FC) producing 5-fluorouracil (5-FU). 5-FU is a common anti-cancer drug that can inhibit DNA synthesis leading to cancer cell death. 5-fluorocytosine can interact with digestive bacteria leading to unwanted side effects for cancer patients. Isocytosine Deaminases (ICD) are enzymes that are of interest in the treatment of cancers using Gene Directed Enzyme Prodrug Therapy (GDEPT). ICDs can deaminate the prodrug 5-fluoroisocytosine (5-FIC) also producing the drug 5-FU.  5-FIC will likely not be recognized by digestive bacteria due to ICDs being less common than CDs resulting in reduced side effects for patients. In this study, the crystal structure of URA4, an isocytosine deaminase, was determined using X-ray crystallography. Using the crystal structure of URA4, ten point mutations were made in the active site to better understand the activity of URA4. Live cell assays have been used to determine the viability of human cancers cells after URA4 and the prodrug 5-FIC were added. The enzyme kinetic characteristics were determined for the mutants using both an absorbance assay and isothermal titration calorimetry assay.

Type

Text

Keywords

Isocytosine Deaminase, Cancer, X-ray Crystallography, Isothermal Titration Calorimetry

Publisher

Western Washington University

OCLC Number

1380913919

Subject – LCSH

Cytosine; X-ray crystallography; Calorimetry; Cancer

Format

application/pdf

Genre/Form

masters theses

Language

English

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

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