Degree Type


Date of Award


Degree Name

Doctor of Philosophy


Biochemistry, Biophysics and Molecular Biology

First Advisor

Richard B Honzatko


Hexokinase I (HKI) is the pacemaker of glycolysis in brain and red bold cells, being subject to potent feedback inhibition by its product glucose 6-phosphate (Glc-6-P) and the relief of that inhibition by inorganic phosphate (Pi) and glucose (Glc). Presented here is a comprehensive model of inhibition and relief of inhibition for recombinant human HKI derived from crystal structures, molecular dynamics simulations, kinetics and directed mutations. Ligand interactions at each half of HKI are comparable in crystal structures, but in simulations, 2-deoxyglucose 6-phosphate (2-deoxyGlc-6-P) and mannose 6-phosphate (Man-6-P) lose hydrogen bonds at the C-terminal half, consistent with early stages of ligand dissociation. 1,5-Anhydroglucose 6-phosphate (1,5-anhydroGlc-6-P), adopts an alternative binding mode, and stabilizes a slightly open C-terminal half. The kinetics of D413N and/or D861N enzymes (D413 and D861 interact with the 1-OH group of Glc-6-P) are consistent only with synergistic binding of two molecules of 1,5-anhydro-Glc-6-P to sites with unequal constants of dissociation. The crystal structure and simulation of HKI with glucose 1,6-bisphosphate (Glc-1,6-P2) implicate Lys418 (N-terminal half) and Lys866 (C-terminal half) in stabilizing inhibited conformations of HKI, a finding support by directed mutations. Pi displaces 1,5-anhydroglucitol 6-phosphate from the N-terminal half, but not from the C-terminal half. Glucose (Glc) up to concentration of 1 mM interacts synergistically with 1,5-anhydroGlc-6-P, but at higher concentrations becomes an antagonist of inhibition. A site-affinity model quantitatively accounts for these observed phenomena in wild-type and mutant forms of HKI. For wild-type HKI, inhibitor binds to sites of unequal affinity, coupled by negative coorperativity, with the site of highest affinity at the N-terminal half. Pi-displaces inhibitor from the N-terminal half, but does not prevent inhibition at the C-terminal half. Potent inhibition requires two molecules of bound glucose to HKI, and the binding of Glc to the active site seems primarily responsible for creating a state of HKI susceptible to potent inhibition. The mapping of mutations that affect allosteric mechanism in HKI clearly define the N-terminal inhibitor pocket and the flexible subdomain as key components of allostery in HKI.


Copyright Owner

Lu Shen



Date Available


File Format


File Size

158 pages

Included in

Biochemistry Commons