Date of Award

2019

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Chemistry

Abstract

Isocitrate dehydrogenase (IDH) is a critical enzyme in the tricarboxylic acid (TCA) cycle and other metabolic processes. IDH catalyzes the oxidative decarboxylation of isocitrate to ⍺-ketoglutarate (⍺-KG) with the reduction of NAD(P)+ to NAD(P)H. Mutations in IDH-1 are found in approximately 80% of grade II-III gliomas and secondary glioblastomas in humans. Mutant IDH-1 is characterized by a gain of function activity where ⍺-KG is reduced to 2-hydroxyglutarate (2-HG) by NADPH. This neomorphic activity results in altered cell metabolism. 2-HG also binds competitively with many α-KG dependent dioxygenases, including some essential histone demethylases. IDH-1 mutations are thought to occur early in, and drive the progression of, cancerogenesis. C. elegans is a popular model organism in biological and biomedical research due to its small size and short life span. About 35% of C. elegans genes have human homologs, including cytosolic IDH-1. The C. elegans IDH-1 enzyme could be a potential model for studying IDH-1 mutations and their effects. Previous research conducted by Bourlett in 2014 resulted in the isolation and kinetic characterization of wild type C. elegans IDH-1. In 2015, Hoyt isolated mutant C. elegans G97N IDH-1 and demonstrated some activity. In this study, C. elegans wild type and G97N mutant IDH-1 genes were overexpressed in Rosetta E. coli. The enzymes were purified using the IMPACT purification system. The concentration of enzyme in the purified fractions was also determined. Isocitrate Michaelis-Menten kinetics curves were obtained for each enzyme. Wild type IDH-1 had a specific activity of 20.8 ± 1.2 μmol min-1 mg-1, a Vmax value of 0.00736 ± 2.3x10-4 μmol/min, and a KM value of 19 ± 2 μM. The G97N IDH-1 had a specific activity of 1.65 ± 0.22 μmol min-1 mg-1, a Vmax value at 0.00076 ± 3.1x10-5 μmol/min, and a KM value of 19 ± 3 μM.

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