Kinetic Characterization of the C. elegans Metabolic Enzyme IDH-1

Noah Bourlett

Date of Award

2014

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

C. elegans, Glyceraldehyde-3-Phosphate Dehydrogenase, Kinetics

Area of Concentration

Biochemistry

Abstract

Isocitrate dehydrogenase is a component enzyme of the tricarboxylic acid cycle which converts isocitrate into α-ketoglutarate with the concomitant reduction of NAD(P)+ to NAD(P)H and release of CO2. Identical point mutations of the human cytosolic NADP-dependent form of this enzyme, IDH1, occur with high incidence in diffuse gliomas and glioblastomas, and recent research has shown that this mutant isocitrate dehydrogenase plays a causative role in tumorigenesis. While research is in progress investigating both wild-type and mutant IDH1 enzymes, the underlying tumorigenic mechanism has yet to be elucidated. C. elegans is a small nematode worm which has become a popular model organism that has been used successfully in medical research as a model of myriad human systems. Investigation of the worm enzyme IDH-1, orthologous to human IDH1, could offer insight into the biological role and tumorigenic mechanism of the human enzyme. Very little research has been done on the worm enzyme, and it has not been kinetically characterized to date. The C. elegans idh-1 gene was inserted into the expression vector pTXB3 (NEB) and overexpressed in E. coli Rosetta cells. The expressed IDH-1 was fused to an intein-chitin binding domain, purified by affinity chromatography via a chitin bead column, and purified by DTT-induced intein self-cleavage. The purified IDH-1 samples were quantitated by a Bradford assay, kinetics assay conditions were optimized, and optimized substrate kinetics curves were obtained. The optimal pH range of C. elegans IDH-1 was determined to lie between pH 7.5-9.5, the KM for isocitrate was 19.0 ± 2.6 μM, the KM for NADP+ was 4.8 ± 0.7 μM, the KM for Mn2+ was 7 ± 1 μM, and the specific activity was calculated to be 25.0 ± 3.8 Units/mg.

This document is currently not available here.

Share

COinS