Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Richard B. Honzatko
Human hexokinase Type-I (HKI) binds to the outer mitochondrial membrane, and in so doing protects the mitochondrion, controls adenine nucleotide flux through the membrane and blocks mitochondrion-linked apoptosis. Adenosine 5’-triphosphate (ATP) releases HKI from the outer mitochondrial membrane, but the mechanism of ATP release is unclear. ATP-release is not due to the generation of glucose 6-phosphate (G6P) from ATP and glucose or by the binding of ATP to HKI at its catalytic or regulatory domains. Instead, the voltage dependent anion channel (VDAC) has a high affinity binding site for ATP as revealed by titrations using ATP and fluorescent analogs of ATP. Single lysine-to-methionine mutations of 15 residues within the pore of VDAC link ATP binding to the NZ atom of Lys256. Giant unilamellar vesicles (GUVs) bind the N-terminal half of HKI only if VDAC is embedded in the vesicle bilayer, demonstrating VDAC alone is sufficient (and necessary) to localize HKI to a membrane. ATP releases the N-terminal half of HKI from VDAC-embedded GUVs, demonstrating ATP-release is due to the binding of ATP to VDAC.
b-Ketoacyl–(acyl-carrier-protein) synthase III (FabH) catalyzes the first condensation reaction of fatty acid biosynthesis. Highly purified FabH from Escherichia coli is unstable at elevated concentrations and precipitates irreversibly from solution. FabH achieves high levels of stability as the acetyl adduct of Cys112, paired with chloride. The resting state in vivo for FabH may be as the chloride-stabilized acetyl adduct of Cys112.
Watanabe, Muneaki, "Role of Non-catalytic Ligands in Macromolecule Function" (2016). Graduate Theses and Dissertations. 15837.