Allosteric Inhibition of Thermus Thermophilus Phosphofructokinase

Abstract

Thermus thermophilus phosphofructokinase (TtPFK) comes from an extreme thermophile and exhibits entropically-driven inhibition by phosphoenolpyruvate (PEP). Interestingly, a PFK from the moderate thermophile Bacillus stearothermophilus also exhibits entropically-driven inhibition, while enthalphically-driven inhibition is observed for PFK from mesophilic E. coli. Although the thermodynamics of inhibition are similar for TtPFK and BsPFK, TtPFK exhibits a much weaker coupling between the inhibitor and substrate (ΔGay = 1.60±0.04 kcal/mol) when compared to that of BsPFK (ΔGay = 5.0±0.9 kcal/mol). Sequence alignment and crystal structures of BsPFK suggest that there is a network of interacting residues leading from the allosteric binding site to the active site. In the apo form, H215 forms a hydrogen bond with T158 and T156 interacts with D12 across the interface. In the inhibitor-bound form, T158 forms a hydrogen bond with D12. In TtPFK these interactions are missing due to substitutions at positions 215 (Ser) and 158 (Ala). Changing the amino acid residues at these positions to the corresponding amino acids in BsPFK resulted in an increase in coupling free energy to ΔGay = 2.47±0.02 kcal/mol for S215H and ΔGay = 2.45±0.04 kcal/mol for A158T. Currently the double mutant A158T/S215H is being investigated to see if the coupling can be augmented to the level of BsPFK coupling. Supported by NIH grant GM33216 and Welch Foundation grant A1548.