This research explores how post-translational modifications (PTMs) like acetylation and phosphorylation regulate key metabolic enzymes, including phosphofructokinase (Pfk), glucokinase (GK), and isocitrate dehydrogenase (IDH). Using genetic code expansion (GCE), a precise method for introducing modified amino acids, I investigated the effects of site-specific PTMs on enzyme activity, substrate binding, and metabolic control. By integrating enzymology, comparative genomics, and cellular models, this study provides insights into how PTMs influence metabolic regulation in prokaryotic and cancer cell systems, with implications for disease mechanisms and therapeutic strategies.