Enhancing Allosteric Response in Thermus Thermophilus Phosphofructokinase

Abstract

Phosphofructokinase (PFK) from an extreme thermophile, Thermus thermophilus (TtPFK), exhibits 17-fold stronger binding to its inhibitor, PEP, and 34-fold weaker coupling between the binding of PEP and substrate, Fructose-6-phosphate (F6P), when compared at 250C to the PFK from another thermophile, Bacillus stearothermophilus (BsPFK). BsPFK is 57% identical in sequence. Since no 3-dimensional structural information is available for TtPFK, we turned to the crystal structures of BsPFK in search for the possible explanation. There is a network of residues, D59, T158, and H215, that leads from the allosteric site to the nearest active site, and that undergoes a significant rearrangement when PEP binds to free enzyme. In the apo form of BsPFK, H215 forms a hydrogen bond with T158. In the inhibitor-bound form, T158 is further removed from the allosteric binding site, and D59 forms a hydrogen bond with H215. In TtPFK these interactions are not possible due to nature of residues at these positions: N59, A158, and S215. We hypothesized that recreating this network of residues would strengthen the coupling between the PEP and F6P binding in TtPFK. Single amino acid substitutions at each of these positions resulted in some increase in binding free energy. The three combinations of double mutations produced a more significant increase in coupling free energy, which appeared in each case to be roughly the sum of the changes in coupling free energy produced by the individual mutants. Interestingly, the level of coupling attained by introducing all three mutations is similar to that seen in BsPFK, and the binding affinity of PEP was weakened to the level exhibited by BsPFK (ΔGay = 3.98±0.03 kcal/mol, ΔGy = −5.61±0.01 kcal/mol). Supported by NIH grant GM33216 and Welch Foundation grant A1548.