4. Discussion
BsPFK is qualitatively very similar, both structurally and functionally, to other prokaryotic PFKs, the canonical example of which is PFK from E. coli (EcPFK). An important difference, however, e one shared with TtPFK e is the response of the allosteric coupling to variations in temperature. As illustrated in Fig. 3, the response of BsPFK to increasing temperature is opposite that of EcPFK. For BsPFK, PEP becomes a better inhibitor at higher temperature (within the range examined) whereas for EcPFK, PEP becomes a weaker inhibitor as temperature is increased. A thermodynamic analysis of these data indicates that sign of the coupling free energy between PEP and Fru-6-P, which is indicative of the binding antagonism between these two ligands, is established by the constituent coupling entropy for BsPFK and instead of the constituent coupling enthalpy as is the case for EcPFK. We have previously suggested that configurational degeneracy may be an important contributor to this dominant entropy term in enzymes like BsPFK [21e23]. It is tempting when considering this possibility to predict that structural perturbations that increase configurational flexibility may have an effect on the strength of allosteric communication that is analogous to that which temperature does; i.e. increasing flexibility might increase the strength of PEP inhibition as does increasing temperature. Indeed, the notion that an allosteric protein transitions between “Relaxed” and “Tense” forms is as old as the first mechanistic explanation of allosteric behavior and the associated 2-state models that followed [34]. The experiments reported herein were performed in an effort to test this idea.
