Enhancing Allosteric Inhibition in Thermus thermophilus Phosphofructokinase

Abstract

The coupling between the binding of the substrate Fru-6-P and the inhibitor phospho(enol)pyruvate (PEP) in phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus is much weaker than that seen in a PFK from Bacillus stearothermophilus. From the crystal structures of Bacillus stearothermophilus PFK (BsPFK) the residues at positions 59, 158, and 215 in BsPFK are located on the path leading from the allosteric site to the nearest active site and are part of the intricate hydrogen-bonding network connecting the two sites. Substituting the corresponding residues in Thermus thermophilus PFK (TtPFK) with the amino acids found at these positions in BsPFK allowed us to enhance the allosteric inhibition by PEP by nearly 3 kcal mol–1 (50-fold) to a value greater than or equal to the coupling observed in BsPFK. Interestingly, each single variant N59D, A158T, and S215H produced a roughly 1 kcal mol–1 increase in coupling free energy of inhibition. The effects of these variants were essentially additive in the three combinations of double variants N59D/A158T, N59D/S215H, and A158T/S215H as well as in the triple variant N59D/A158T/S215H. Consequently, while the hydrogen-bonding network identified is likely involved in the inhibitory allosteric communication, a model requiring a linked chain of interactions connecting the sites is not supported by these data. Despite the fact that the allosteric activator of the bacterial PFK, MgADP, binds at the same allosteric site, the substitutions at positions 59, 158, and 215 do not have an equally dramatic effect on the binding affinity and the allosteric activation by MgADP. The effect of the S215H and N59D/A158T/S215H substitutions on the activation by MgADP could not be determined because of a dramatic drop in MgADP binding affinity that resulted from the S215H substitution. The single variants N59D and A158T supported binding but showed little change in the free energy of activation by MgADP compared to the wild type TtPFK. These results support previous suggestions that heterotropic inhibition and activation occur by different pathways prokaryotic PFK.

Turning up the heat mimics allosteric signaling in imidazole-glycerol phosphate synthase

“…Temperature plays a crucial role in allosteric mechanisms 29 – 32 , especially in thermophilic enzymes that remain active at elevated temperatures. The elevation of temperature has been linked in some K-type enzymes 30 to an inversion of allosteric modulation and in others to an enhancement of modulation 32 , 33 . In all these cases, the temperature-induced effects are a consequence of an adjustment in substrate affinity upon allosteric ligand binding.…”

Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

“…In this view, it has been shown that allosteric potential can be decreased by using mutagenesis to disrupt specific interactions between fructose-1,6-bisphosphatase and its effector, adenosine monophosphate (33). Recent studies have also shown that engineering can increase allosteric potential, enhancing inhibition of phosphofructokinase by phosphoenolpyruvate (34). An allosteric potential can even be engineered into enzymes where previously one did not exist (35).…”

Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble

“…Many studies on allostery have focused on the importance of discrete networks of residues that are involved in signal propagation upon effector binding. ,, Such networks have also been identified in the St TrpS complex, where remote mutations can shift the ensemble to favor open conformational states by disrupting hydrogen bonds or salt bridges that form upon COMM domain closure, thereby impeding catalysis. Mutations at the subunit interface can be similarly disruptive .…”