| dc.description.abstract | Phosphofructokinase from Bacillus stearothermophilus (BsPFK) and Escherichia coli (EcPFK) are allosterically inhibited by downstream glycolytic pathway intermediate phosphoenol-pyruvate (PEP). The coupling free energy, ∆Gay, describes the interaction between substrate, Fru-6-P, and PEP. Positive for inhibition, ∆Gay is the standard free energy for the following disproportionation equilibrium:
PEP:PFK + PFK:Fru-6-P ⟷ PFK + PEP:PFK:Fru-6-P
and quantitatively expresses the nature and magnitude of the allosteric effect. This reaction provides the key to understanding why ∆Gay achieves its particular value. For EcPFK, the larger positive enthalpy term determines the positive sign for ∆Gay. In BsPFK both components are negative, and the larger absolute value of the entropy term drives inhibition, suggesting fundamentally different mechanisms may transfer the allosteric signal between PEP and Fru-6-P binding sites. In this body of work, BsPFK and EcPFK structures are perturbed by both urea and engineered “holes”, and the effect on the poise of the equilibrium in response is examined. Methyl-TROSY NMR was also used to obtain structural information on all four species of BsPFK that contribute to the equilibrium.
Both activity and ∆Gay increase at low urea concentrations for BsPFK, but not for EcPFK. Van’t Hoff analysis on BsPFK indicates that the absolute values for both the entropy and enthalpy components of ∆Gay change, and that the increase in allosteric coupling results from a larger change in entropy. Two of the three hole mutations had significant, but opposite, effects on the coupling. A 4-fold augmentation of coupling was seen for I153V-BsPFK, and the larger effect was determined to be on the enthalpy component. An analogous mutation in EcPFK, L154V, served as a control and had a minimal effect on the coupling. Methyl-TROSY NMR was used to probe structural changes throughout BsPFK related to allosteric coupling, and it was shown that there is not likely a discrete pathway involved in the propagation of the allosteric signal. Instead, residues throughout the enzyme are identified as contributors to the allosteric coupling. Together with the urea and hole mutant results we paint a picture of allosteric coupling in BsPFK that involves global changes in both the conformation entropy and structure. | en |
