Date of Award

2024

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Biochemistry, Neuroscience

Abstract

The metabolic enzyme isocitrate dehydrogenase (IDH-1) is the C. elegans ortholog of human cytosolic IDH1. The mechanism for both the human and the C. elegans IDH1 enzymes reversibly convert the substrate isocitrate (ICT) to α-ketoglutarate (αKG). The IDH-1 enzyme is particularly interesting due to the association of certain IDH1 mutations to human cancers. The mutations that are associated with oncogenesis are shown to inhibit the activity of IDH1 by introducing a neomorphic activity that converts αKG to α-hydroxyglutaric acid, also called 2- hydroxyglutarate (2HG).To determine if IDH-1 may be a reliable model system to explore the cellular abnormalities produced by IDH-1 mutations, three variants of the C. elegans IDH-1 enzyme were analyzed; Wild-type IDH-1 as well as two mutants, G98N and R133H, which correspond to the human G97N and R132H mutations, respectively. Each of the three variants of the C. elegans IDH-1 gene was inserted into an expression vector pET303 (NEB) and overexpressed in E. coli Rosetta cells. The expressed IDH-1 enzyme was fused to an intein-chitin binding domain. It bound to a chitin bead column and was released by DTT-induced intein selfcleavage. Bradford assays were used to quantitate the purified IDH-1 samples. The initial characterization of wild-type IDH-1 using UV-Vis spectroscopic assays to detect NADPH led to the discovery of increased enzyme activity in the presence of Mn2+ and NADP+ compared to Mg2+ and NAD+, respectively. The experimental KM for αKG was lower (14 ± 2 μM) for the C. elegans R133H mutant when compared with the αKG KM values previously reported for the human R132H mutant. The experimental results indicated that the wild-type IDH-1 and R133H mutant had similar characteristics in the forward reaction to the human wild-type IDH1 and its R132H mutant, respectively. In contrast, the G98N mutant had a higher forward enzyme activity than the human G97N mutant. Additional analysis via liquid chromatography-mass spectrometry (LC-MS) was used to measure the amount of 2HG produced by the C. elegans enzymes in the reverse reactions. The LC-MS results indicated that the R133H mutant produced more 2HG per reaction (80 ± 20 2HG/ICT) than the G98N (13 ± 1 2HG/ICT) and wild-type (1.5 ± 0.3 2HG/ICT) enzymes. The buildup of 2HG in the system is associated with an increased risk of oncogenesis, indicating that the human ortholog to the C. elegans R133H mutant may pose an increased risk of oncogenesis compared with the wild-type and the G97N mutant.

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