Abstract
An investigation into the kinetics and regulatory properties of the type-1 phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus (TtPFK) reveals an enzyme that is inhibited by PEP and activated by ADP by modifying the affinity exhibited for the substrate fructose 6-phosphate (Fru-6-P) in a manner analogous to other prokaryotic PFKs. However, TtPFK binds both of these allosteric ligands significantly more tightly than other bacterial PFKs while effecting a substantially more modest extent of inhibition or activation at 25 °C, reinforcing the principle that binding affinity and effectiveness can be both independent and uncorrelated to one another. These properties have allowed us to establish rigorously that PEP only inhibits by antagonizing the binding of Fru-6-P and not by influencing turnover, a conclusion that requires kcat to be determined under conditions in which both inhibitor and substrate are saturating simultaneously. In addition, the temperature dependence of the allosteric effects on Fru-6-P binding indicate that the coupling free energies are entropy-dominated, as observed previously for PFK from Bacillus stearothermophilus but not for PFK from Escherichia coli , supporting the hypothesis that entropy-dominated allosteric effects may be a characteristic of enzymes derived from thermostable organisms. For such enzymes, the root cause of the allosteric effect may not be easily discerned from static structural information such as that obtained from X-ray crystallography.