Author ORCID Identifier:
Date of Graduation
5-2026
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Chemistry (PhD)
Degree Level
Graduate
Department
Chemistry & Biochemistry
Advisor/Mentor
Fan, Chenguang
Committee Member
Dong, Bin
Second Committee Member
Lewis, Jeffrey
Third Committee Member
Adams, Paul
Keywords
Bacterial Glucokinase Inactivation; Lysine Modification; Chemical Inhibition, GLK inhibitors
Abstract
Glycolysis and related metabolic pathways are controlled by various post-translational modifications (PTMs) that affect enzyme activity and metabolic flow. In E. coli, glucokinase (GLK) sits at the crossroads of glycolysis and the pentose phosphate pathway and carries out the first committed step of glycolysis: ATP-dependent phosphorylation of glucose. While it's known that glucose binding causes GLK to close its domains, how phosphate binding, lysine modification, and small-molecule inhibitors regulate GLK's shape and function is still not fully understood. Here, I combine structural biochemistry and enzymology to figure out how phosphate binding, specific mutations, and chemical inhibitors affect E. coli GLK. We solved the crystal structures of phosphate-bound GLK (PDB 9DUC) and the phosphate–glucose complex (PDB 9DV). Phosphate binds near the glucose pocket and stabilizes several key residues. Comparing these structures shows that phosphate makes parts of the enzyme less flexible, especially in certain helices and loops. When glucose is present, specific residues move inward, causing the enzyme to close up. Overall, phosphate helps get the enzyme ready for glucose binding by making it more stable and primed for closure. To see how specific lysine residues, affect GLK, I looked at two mutants: K214Q and K216Q (PDB 9YRS and 9PYU). Both still bind phosphate normally, but K214Q causes big changes to the enzyme's backbone and makes some regions more disordered. This mutant pushes glucose-binding residues outward, making it harder for the enzyme to close. K216Q, on the other hand, doesn't disrupt things as much. This shows that these two lysines have different roles: K214 helps stabilize GLK for glucose binding and closure, while K216 is less critical. This helps explain how lysine acetylation might tweak enzyme activity by shifting its shape rather than directly affecting the catalytic site. I also solved the structure of GLK bound to the glucose analog D(+)-glucosamine (PDB 10QN). Even though glucosamine sits in the glucose pocket, it doesn't make the enzyme close up like glucose does. Key residues are pushed outward, and the enzyme stays more flexible. Activity assays show that glucosamine inhibits GLK in a dose-dependent way, with an IC₅₀ around 19 mM. So, just having something in the binding pocket isn't enough to turn the enzyme on. In summary, this work shows how phosphate, glucose, mutations, and inhibitors each shape the structure and activity of E. coli GLK. By mapping out these effects, I hope to make it easier to engineer metabolism or develop new GLK inhibitors in the future.
Citation
Andrews, J. R. (2026). Mechanisms of Bacterial Glucokinase Inactivation through Site-Specific Lysine Modification and Chemical Inhibition. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/6156
