Date Thesis Awarded

5-2017

Document Type

Honors Thesis

Degree Name

Bachelors of Science (BS)

Department

Chemistry

Advisor

Dr. Lisa Landino

Committee Members

Dr. Christopher Abelt

Dr. Douglas Young

Abstract

Reactive oxygen species (ROS) play a critical role in intracellular signaling mechanisms. These small molecules initiate reversible, post-translational modifications of redox-sensitive proteins. Pyruvate kinase (PK) is a key glycolytic enzyme whose activity is modulated by ROS; oxidation inhibits its catalytic function through a reaction at a critical cysteine near the active site. PK’s central role in moderating glycolytic flux makes it highly regulated through both structural and allosteric effectors. This research explores the extent of cysteine modification of PK under various models of intracellular conditions. PK was treated with oxidants and allosteric regulators, and changes in the enzyme’s activity and cysteine reactivity were monitored through spectrophotometric and SDS-PAGE assays. Addition of oxidants decreased both PK activity and cysteine accessibility. Addition of ATP and fructose-1,6-bisphosphate (FBP) protected cysteines from tagging and oxidation. Other allosteric effectors like PEP had no effect on cysteine accessibility at low concentrations. Quantifying the reactivity of PK’s cysteines allows researchers to further understand the nuanced relationship that exists between intracellular redox conditions and protein activity. This research has been completed in tandem with Julia Zuercher.

Included in

Biochemistry Commons

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