Date of Award

1991

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Ezzat S. Younathan

Abstract

The glycolytic pathway enzyme phosphofructokinase (E.C. 2.7.1.11) has as its substrate and activator two different carbohydrate phosphate esters. Both are derivatives of D-fructose in the furanose ring form. This dissertation is divided into two chapters: the first deals with the conformational specificity of the active site and the second the structure of the activator. The compound 2,5-anhydro-3,4-O-(1,2-ethanediyl)-D-mannitol was designed to test the active site. The synthesis utilized a phase-transfer cyclo-dialkylation of a vicinal diol to yield a trans-fused 2,5,8-trioxabicyclo (4.3.0) nonane system. The final product was shown to exist in a locked conformation in solution by temperature dependent n.m.r. experiments which showed no line shape change. The data indicate that the five-membered ring is locked by the trans-fused six-membered 1,4-dioxane ring into a twist $\sp4$T$\sb3$ conformation. A single crystal X-ray study was carried out. The crystalline product exists in an ideal twist conformation with a pseudorotation angle of 0$\sp\circ$amplitude of 47.2$\sp\circ$in agreement with the n.m.r. results. The compound, as the monophosphate, is intended to verify the linear plot of substrate efficacy index versus $\beta$-$\sp4$T$\sb3$ concentration observed with several ketose 6-phosphates. Chapter two describes detailed n.m.r. studies of the activator molecule, D-fructofuranose 2,6-bisphosphate, which allowed the unequivocal assignment of all the proton, carbon and phosphorus resonances. Several unexpected chemical shift values and coupling constants were obtained. The usual near-gauche orientations of C-1 and C-3 to P-2, obtained by molecular mechanics calculations, can explain their small vicinal coupling constants in contrast to the expected larger value seen for C-5 to P-6. Reduction did not affect the n.m.r. spectrum substantiating that C-2 is phosphorylated. Oxidation yielded an unstable intermediate which decomposed by a beta elimination mechanism involving the phosphate group on C-6. These data establish unequivocally the $\sp1$H, $\sp{13}$C and $\sp{31}$P assignment and explain the observed anomalous shifts. Moreover, they establish that the activator of fructose 6-phosphate 1-kinase is the $\beta$-anomer of the $\sp4$T$\sb3$ conformer of D-fructose 2,6-bisphosphate. The conclusion from both investigations is that both the active site and the activation site of phosphofructokinase seem to prefer the $\sp4$T$\sb3$ conformer of these fructofuranose ligands.

Pages

63

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