Date of Award

1991

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

M. D. Barkley

Abstract

Phosphofructokinase catalyzes the phosphorylation of $\beta$-fructose 6-phosphate (Fru-6-P) to $\beta$-fructose 1,6-bisphosphate. The first part of this dissertation shows that phosphofructokinase from Bacillus stearothermophilus (Bs-PFK) is a tetramer in the region pH 4.5-9.5 unlike the enzymes from mammalian sources, which exhibit association-dissociation reactions. Circular dichroism, steady-state fluorescence, and static and dynamic light scattering were used to study the conformational properties of the enzyme. Inactivation of the enzyme below pH 7.0 is not due to dissociation of the tetramer to dimers or monomers. In the second part, the fluorescence of the lone tryptophan was investigated by steady-state and time-resolved techniques. The decay of Bs-PFK can be best described as a discrete double exponential with lifetimes of $\sim$1.6 and 4.4 ns. The decay-associated emission spectra of the two components are identical. Similar results were obtained in D$\sb2$O, suggesting that the heterogeneous emission is not due to excited-state proton transfer. The activation energy for the temperature-dependent nonradiative decay rate was $\sim$0.94 Kcal/mol. The emission had a quantum yield of 0.30 $\pm$ 0.03 with a rate constant for acrylamide quenching, 0.62 $\times$ 10$\sp9$ M$\sp{-1}$ s$\sp{-1}$ which is 4-fold lower than the value expected for a diffusion controlled reaction. The fluorescence anisotropy is 0.18 suggesting that the tryptophan environment is fairly rigid. From the anisotropy decay measurements, a single exponential decay with rotational correlation time of $\sim$40 ns with r(0) value of $\sim$0.19 was obtained. Finally, the effects of allosteric ligand binding and site-specific mutation on the conformation of Bs-PFK were studied. Addition of adenosine triphosphate (ATP) or Fru-6-P did not cause any change in fluorescence. However, there is about 7% decrease of quantum yield and 2 nm red shift of emission maximum, upon adding the inhibitor PEP. Fru-6-P restores the fluorescence parameters to those of native Bs-PFK indicating the substrate induced allosteric transition from T- to R-conformation. The Arg-252 to Ala-252 mutation caused opposite effects: increase of quantum yield and 2 nm blue shift of emission maximum.

Pages

150

Share

COinS