Allosteric Regulation in Phosphofructokinase from the Extreme Thermophile Thermus thermophilus

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 do 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 rigorously establish that PEP only inhibits by antagonizing the binding of Fru-6-P, and not by influencing turnover – a conclusion that requires kcat 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 B. stearothermophilus but not for PFK from E. 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 might be obtained from X-ray crystallography.

Carbohydrate Kinases: A Conserved Mechanism Across Differing Folds

“…Phosphofructokinases are amongst the best understood allosterically regulated proteins. Both prokaryotic [66,69] and eukaryotic [70] PFKs show allosteric regulation to feedback through glycolysis. Indeed, eukaryotic PFKs have over 20 known effectors and two effector binding sites [70].…”

Enhancing Allosteric Inhibition in Thermus thermophilus Phosphofructokinase

“…An increase in the homotropic cooperativity with the addition of PEP was seen in all mutants, similar to what is seen in wild type TtPFK. 1 …”

“…The pALTER plasmid with the wild type TtPFK gene was used as the starting template for mutagenesis. 1 For double and triple substitutions, the plasmid containing the gene with the single or double was used as a template. The mutations were introduced using QuikChange (Stratagene, La Jolla, CA) using a pair of complementary primers.…”

“…Phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus (TtPFK) is allosterically inhibited by phospho( enol )pyruvate (PEP) and activated by MgADP, 1 as are PFKs isolated from virtually all other prokaryotic organisms. These effects are manifested by a change in the affinity for the substrate, fructose 6-phosphate (Fru-6-P), upon binding of the allosteric ligand.…”

“…However, PEP inhibits BsPFK 30-fold more strongly, while MgADP activates both enzymes with nearly equal effectiveness. 1 In an attempt to pinpoint the basis of the weaker coupling between PEP and Fru-6-P exhibited by TtPFK, we have analyzed the available structures, determined by X-ray crystallography, of BsPFK in the apo form, 4 BsPFK bound to the inhibitor phosphoglycolate, 5 and BsPFK bound to both the substrate, fructose 6-phosphate (Fru-6-P) and the allosteric activator (ADP). 6 From these structures one can identify a series of residues involved in an extensive hydrogen-bonding network that extends from the allosteric site to the closest active site.…”