Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Herbert J. Fromm
Fructose-1,6-bisphosphatase (FBPase) catalyzes the reaction of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. The investigations described here concern the structure and function of FBPase, with special focus on those properties relating to the allosteric regulation of catalysis. It is our hypothesis that a loop spanning residues 52 through 72 plays a crucial role in the mechanism of allosteric regulation. Testing this hypothesis, a fluorescent reporter group was incorporated into loop 52-72, confirming the existence of three states of the loop dependent on the state of ligation: disordered, disengaged, and engaged. Furthermore, restriction of backbone flexibility by replacement of two residues located in the hinge of loop 52-72 with proline broadly effects catalysis and the mechanism of AMP regulation. The N-terminal region of the enzyme assists in stabilizing the loop 52-72 in this region of the protein in the disengaged state. Deletion of the first ten residues or specific point mutations dramatically alters the mechanism of allosteric regulation. A model describing the relative free energies associated with the three states of loop 52-72 was generated to account for the functional properties of wild-type and mutant enzymes displaying biphasic inhibition by AMP. To further describe the mechanism of AMP inhibition, a method for producing and isolating hybrid enzymes was developed. Spontaneous subunit exchange was used to create hybrids between wild-type enzyme and a mutant with disrupted AMP-binding sites. The hybrid enzymes having different arrangements of two subunits either capable or incapable of binding AMP, reveal a specific pathway for AMP binding to the tetramer. Overall, the work presented here is a significant advancement in the understanding of the fundamental mechanisms of allosteric regulation of FBPase.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Scott William Nelson
Nelson, Scott William, "Allosteric regulation of fructose-1,6,-bisphosphatase " (2002). Retrospective Theses and Dissertations. 398.