Date of Award

5-2014

Document Type

Campus Access Thesis

College

College of Theology, Arts, & Sciences

Department

Math & Science

Degree Name

Chemistry, BA

First Supervisor

Dr. Michael Godsey

Keywords

deoxycytidine kinase, deoxycytidine triphosphate, cytosine arabinoside

Abstract

Deoxycytidine kinase (dCK) is responsible for catalyzing the phosphorylation of nucleosides to monophosphate forms within the nucleoside salvage pathway by using nucleotide triphosphates ATP or UTP as a phosphoryl donor. Several important antiviral and anticancer nucleoside prodrugs are phosphorylated by dCK including cytosine arabinoside (Ara-C). The phosphorylation of Ara-C to its monophosphate form is considered rate-limiting in its activation. The activated form of this prodrug, Ara-CTP, blocks DNA synthesis, resulting in leukemic cell death. dCK also has a role in immune function, since dCK inhibitors are potential treatments for immune disorders. Wild-type dCK has been reported to be competitively inhibited by deoxycytidine triphosphate (dCTP). This study was conducted in order to determine whether several types of mutant dCK would exhibit a different kind of inhibition or be uninhibited with dCTP compared to wild-type. dCK residues Asp241 or Phe242 were altered in the phosphoryl donor binding loop by site-directed mutagenesis. Expression of wild-type and mutated histidine- tagged dCK was done in E. coli. dCK was purified to >95% purity by immobilized metal affinity chromatography (IMAC) with Co2+ resin. SDS-PAGE gel electrophoresis was utilized to analyze protein expression and purification. Kinetic assays were done with ATP concentrations varying from 5 µM to 500µM. To test for inhibition of dCK, 10µM dCTP was added. Over time, oxidation of NADH was monitored by UV/Vis spectroscopy. Kinetic properties for dCK were calculated from the raw data with Logger Pro. We determined that D241N, D241N/F242L, and F242L dCK all had competitive inhibition similar to wild-type with respect to dCTP.

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