Degree Type

Dissertation

Date of Award

1985

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Abstract

The fructose 1,6-bisphosphatase (FBPase) reaction was investigated in the reverse direction by using fructose 2,6-bisphosphate (2,6FBP). The effector was found to be a potent inhibitor of the reverse reaction substrates fructose 6-phosphate (F6P) and inorganic phosphate (P(,i)). Inhibition of bovine liver FBPase by 2,6FBP was competitive with respect to both substrates, and slope replots were linear. In the context of other accumulated kinetic data, the results serve to support a Random Bi Uni mechanism as the most likely mechanism for the reverse reaction. In addition, two models consistent with the data are presented for the interaction of 2,6FBP with FBPase;The interaction of 2,6FBP and adenosine-5'-monophosphate (AMP) with bovine liver FBPase was investigated further by using ('1)H and ('31)P nuclear magnetic resonance (NMR). Mn('2+) bound to FBPase was used as a probe to map the active and AMP allosteric sites of the enzyme. The distances between enzyme-bound Mn('2+) and the phosphorus atoms at the C-6 positions of F6P, (alpha)-methyl D-fructofuranoside 1,6-bisphosphate and 2,6FBP were very similar. Likewise, the enzyme-Mn to phosphorus distances obtained for inorganic phosphate, the C-1 phosphorus atom of (alpha)-methyl D-fructofuranoside 1,6-bisphosphate and the C-2 phosphorus atom of 2,6FBP were nearly identical, suggesting that 2,6FBP binds at the active site of FBPase;On the other hand, the distance between enzyme-bound Mn('2+) and the phosphorus atom of AMP was significantly shorter than the distances obtained for any of the aforementioned ligands, but the presence of 2,6FBP caused the enzyme-Mn to phosphorus distance for AMP to lengthen markedly. In addition, binding studies showed that 2,6FBP enhances the binding of AMP to FBPase, indicating that 2,6FBP strongly affects the interaction of AMP with the allosteric site of the enzyme;In addition, NMR line broadening of AMP protons was studied at various temperatures. The dissociation rate constant was found to be greater than 20 sec('-1), and the energy of activation for the interaction of the nucleotide with the enzyme was calculated. The presence of 2,6FBP caused the proton resonances to narrow markedly, indicating that 2,6FBP may perturb the exchange between adenosine-5'-monophosphate and fructose 1,6-bisphosphatase.

DOI

https://doi.org/10.31274/rtd-180813-5906

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Nancy Joan Ganson

Language

en

Proquest ID

AAI8514400

File Format

application/pdf

File Size

131 pages

Included in

Biochemistry Commons

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