Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Herbert J. Fromm
Richard B. Honzatko
Type I hexokinase (HKI) is the pacemaker of glycolysis in the brain and red blood cell. Recent work has tied hexokinase association with the mitochondrial membrane to the prevention of apoptosis. With increased hexokinase activity and expression in tumor cells, the importance in understanding the release of HKI from the membrane has become extremely important. Glucose 6-phosphate and nucleotides have been shown to effectively release HKI from the membrane; however, the mechanisms by which these molecules can induce release have never been studied;Expressed recombinant HKI protein was lacking the N-terminus that is required for binding to the mitochondrial membrane. A new construct with a ten histidine tail at the C-terminus was created and found to have an intact N-terminus. Glucose 6-phosphate, a product and potent inhibitor of HKI, has been shown to bind to two different sites on the protein, but the inhibitor binds to only one site under physiological conditions in solution kinetic experiments. A series of site-directed mutations aimed at removing the ability of HKI to bind glucose 6-phosphate were made. After analysis, it has been determined that the N-terminal half glucose 6-phosphate binding site of HKI is responsible for releasing HKI from the mitochondria;Chapter III addresses the topic of ATP-induced release of HKI from the mitochondrial membrane. X-ray crystallography revealed a nucleotide binding site near the N-terminal binding helix of hexokinase I. A similar set of mutations found in Chapter II along with mutations in the nucleotide binding site, were used to identify the site responsible for nucleotide-induced release from the mitochondria. However, it was determined that ATP appears to be binding to porin or the mitochondrial membrane and not HKI;Chapter IV contains a series of solution kinetic experiments were conducted to assign a role to the N-terminal nucleotide binding site. Forty years ago, ADP was shown to be a noncompetitive inhibitor relative to ATP. The allosteric site responsible for causing the mixed inhibition was never identified. Using site-directed mutations of HKI illustrated in Chapter III, the nucleotide site near the N-terminus was indeed found to be the allosteric nucleotide binding site.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
David Andrew Skaff
Skaff, David Andrew, "Release of human brain hexokinase from the mitochondrial membrane " (2006). Retrospective Theses and Dissertations. 3079.