Date of Award

2016

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Chemistry

Abstract

Isocitrate dehydrogenase (IDH-1) is an enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG), a reaction that occurs in the tricarboxylic acid (TCA) cycle. Several mutated forms of the IDH-1 gene have been observed in various forms of cancer. Mutant IDH-1 converts α-KG to 2-hydroxyglutarate (2-HG), which acts as a competitive inhibitor of α-KG-dependent dioxygenases and disrupts normal cell metabolism. The C. elegans IDH-1gene has never been used as a model when studying these mutated isocitrate dehydrogenase genes. Because the C. elegans and human IDH-1 genes are orthologous, the C. elegans gene has the potential to be utilized when researching both the underlying mechanisms of the mutations as well as potential targeted therapies for patients expressing these mutations. In this study, the C. elegans R132H and G97N mutant IDH-1 genes were inserted into the expression vector pTXB3 and overexpressed in Rosetta E. coli. The G97N mutant IDH-1 enzyme was sequenced, tested for solubility, and purified. It was found that the G97N mutant and purified wild-type IDH-1 both had similar rates of activity for the forward reaction. However, the G97N mutant IDH-1 enzyme’s ability to perform the α-KG to 2-HG side reaction was not determined due to time constraints.

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