Seth Hale

Date of Award

2020

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Biochemistry

Abstract

Isocitrate dehydrogenase is a crucial enzyme for the citric acid cycle (CAC) and performs the second step which converts isocitrate dehydrogenase to α-ketoglutarate (AKG). IDH-1 is a cytosolic isoform of isocitrate which has been shown to perform both the forward and reverse reaction. Mutants of IDH-1, namely the R132H and G97N mutants, can convert α-ketoglutarate to the oncometabolite 2-hydroxyglutarate. These two mutants are known oncoproteins and are shown to occur in ~80% of WHO grade II and III gliomas. Several theses have been done on wild type C. elegans IDH-1 and the G97N mutant, with most of these theses being focused on characterizing the steady-state kinetics of both wild type IDH-1 and the G97N mutant. Little research has been done on modeling C. elegans IDH-1 and its mutants in this lab prior to this thesis. This thesis looks to contribute to the steady-state kinetics of wild-type IDH-1, the creation of the R133H mutant, and to the modeling of wild-type, G98N, and R133H C. elegans IDH-1. The creation of the R133H mutant of IDH-1 entailed completing several PCRs to create fragments that would be joined via NEBuilder Hi-Fi assembly. PCR of both fragments was successfully performed and verified via gel electrophoresis. The gel electrophoresis showed two fragments at 5 kb and 3 kb that signify the set B and set A fragments respectively. The subsequent Hi-Fi assembly, however, was found to be unsuccessful via restriction enzyme digests on the resulting plasmids that indicated improper annealing of the PCR products. Kinetic assays were completed on wild type IDH-1 in the presence of 1 mM ATP and 1 mM succinate. The three different reaction mixtures were found to have rates of 0.00325 abs/sec for the wild type, 0.00160 abs/sec for the wild type with the addition of 1 mM ATP, and 0.00481 abs/sec for the wild type with the addition of succinate. These rates show that succinate acts as an activator for IDH-1 and that ATP acts as an inhibitor. Modeling was completed using the SWISS-MODEL which is a program that generates structures of input amino acid sequences based on known structures of homologous proteins. Modeling was completed on the R133H and G98N mutants as well as on wild type IDH-1 for comparison. The histidine in the R133H mutant was shown to not have as deep a reach into the NADP+ binding site as arginine in the wild type. This can lead to decreased interaction with the NADP+ by the histidine as compared to the arginine in wild type IDH-1. The asparagine in the G98N mutant also is more sterically restrictive than the glycine in wild type IDH-1. The inability of asparagine to rotate in the same manner as glycine could impact the binding site for NADP+ and lead to a more favorable state with respect to the NADP+ bound and free IDH-1 states.

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