Date of Graduation

5-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Fan, Chenguang

Committee Member

Sakon, Joshua

Second Committee Member

Du, Yuchun

Third Committee Member

Dong, Bin

Keywords

Acetylation; Enzymology; Genetic Code Expansion; Molecular Biology; Phosphorylation; Post translational modification

Abstract

All living things depend on glycolysis and the TCA cycle for energy production and metabolic control. Post-translational modifications (PTMs) such as acetylation and phosphorylation, which can change enzyme activity, substrate binding, and overall metabolic balance, tightly regulate these pathways. Diseases like cancer and metabolic problems are associated with disruptions in these alterations.

Serine phosphorylation and lysine acetylation are two of the many PTMs that are evolutionarily conserved processes that alter protein function. Previously thought to be a static histone alteration, acetylation is now understood to be a dynamic regulator of metabolic enzymes that modifies contacts, charge, and conformation. In response to biological signals, phosphorylation, a well-known signaling switch, precisely regulates the activity of enzymes. Nevertheless, little is known about the functional effects of site-specific PTMs on metabolic enzymes, specifically how they affect catalytic efficiency, allosteric regulation, and the development of disease. Due to their incapacity to replicate steric or electrostatic disturbances, conventional methods like mutagenesis frequently fall short in reproducing PTM effects. Innovative techniques such as genetic code expansion (GCE) have been developed to address this, allowing for the site-specific inclusion of phosphoserine or acetyllysine through stop codon suppression. This method makes use of pyrrolysyl-tRNA synthetase/tRNA pairs to precisely examine the structural and functional effects of acetylation in vivo. In this research, I used a multidisciplinary method that combines comparative genomics, enzymology, and GCE to analyze how phosphorylation and acetylation rewire the activity of three key enzymes: phosphofructokinase (Pfk), glucokinase (GK), and isocitrate dehydrogenase (IDH). I clarify how site-specific changes affect enzyme kinetics, substrate binding, and metabolic flux by investigating phosphorylation in cancer metabolism using human epithelial cell lines and E. coli as a model for prokaryotic PTM control.

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