Date Thesis Awarded

5-2017

Access Type

Honors Thesis -- Access Restricted On-Campus Only

Degree Name

Bachelors of Science (BS)

Department

Chemistry

Advisor

Lisa Landino

Committee Members

Douglas Young

Mark Forsyth

Randolph Coleman

Abstract

Pyruvate kinase (PK), an important glycolytic enzyme that catalyzes the final step of glycolysis through an irreversible mechanism to produce energy for the cell, contains nine cysteines, some of which are highly reactive and critical for function. This research sought to develop a protocol to measure quantitatively the functionality of PK under different experimental conditions. Using a three stage, consistent procedure, it was possible to analyze the effects of thiol modification by Ellman’s Reagent (DTNB) and oxidation by hypochlorous acid (HOCl) on PK activity. Results showed that both DTNB and HOCl inhibited activity at high concentrations, which illustrates that some cysteines are essential for PK to catalyze its reaction. Furthermore, analysis of cysteine labeling with 5-iodoacetamidofluorescein on SDS-PAGE demonstrates that not all cysteines must be modified to see inhibitory effects. Additionally, incubation studies with ATP and fructose-1,6-bisphosphate, known regulators with different binding sites in PK, located important cysteines in the enzyme and explored their reactivity and significance. This characterization of the enzyme has notable implications in vivo in that PK could potentially serve a protective role for other cysteine-containing proteins necessary for cellular function that might be more sensitive to the effects of oxidative damage, such as tubulin, tau, or other glycolytic enzymes, accumulated damage to which could lead to neurodegenerative diseases, such as Alzheimer’s disease (AD).

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