Madhavi Baiju

Date of Award

2018

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Chemistry

Abstract

Isocitrate Dehydrogenase 1 (IDH1) is an enzyme found in the second step of the citric acid cycle which converts isocitrate to alpha-ketoglutarate, with the reduction of NADP+ to NADPH and the release of CO2. Mutations of the IDH1 enzyme have been found to further tumorigenesis that leads to fatal and belligerent tumors in the brain called glioblastomas. Investigation of IDH1 in C. elegans offers a simple biological model which has the potential to lead to an understanding of what can affect enzyme activity as well as how the wild-type activity compares to the activity of any mutant forms. Additives that have been shown to have activating effects on IDH1 in other species include succinate, ADP, glutamate and NaCl. Those that showed inhibitory effects were oxaloacetate, citrate and KCl. In this study, previous samples of wild-type IDH1 and G97N mutant IDH1 from C. elegans were tested for activity and were found to have minimal activity. Both enzymes were re-purified from frozen overexpression samples, run on SDS-PAGE gels and tested again for activity. The re-purified wild-type IDH1 wild-type showed high activity and was therefore kinetically characterized and used to test the activating and inhibitory effects of various additives. The newly purified IDH1 wild-type enzyme was found to have a KM of 9 ± 2 μM and a VMAX of 0.030 ± 2 x 10-3 μM/min. The only additive to have shown activating effects on IDH1 was 1 mM succinate at 7% stimulation, which was not deemed significant. ADP, although reported to be an activator, was found to have the highest inhibition of IDH1 of 82% at 1 mM. Citrate was found to have the next highest inhibition, showing 61% at 1 mM, followed by oxaloacetate at 20% inhibition at 1 mM concentration. Glutamate, NaCl at 100 mM, and both concentrations of KCl showed to have slight inhibition of IDH1, while NaCl at 50 mM concentration showed no effect on the enzyme.

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