دانلود رایگان مقاله اثر معرفی حفره کوچک بر مهار آلوستریک فسفوفروکتوکیناز

Abstract

The allosteric coupling free energy between ligands fructose-6-phosphate (Fru-6-P) and phospho(enol)pyruvate (PEP) for phosphofructokinase-1 (PFK) from the moderate thermophile, Bacillus stearothermophilus (BsPFK), results from compensating enthalpy and entropy components. In BsPFK the positive coupling free energy that defines inhibition is opposite in sign from the negative enthalpy term and is therefore determined by the larger absolute value of the negative entropy term. Variants of BsPFK were made to determine the effect of adding small cavities to the structure on the allosteric function of the enzyme. The BsPFK Ile → Val (cavity containing) mutants have varied values for the coupling free energy between PEP and Fru-6-P, indicating that the modifications altered the effectiveness of PEP as an inhibitor. Notably, the mutation I153V had a substantial positive impact on the magnitude of inhibition by PEP. Van't Hoff analysis determined that this is the result of decreased entropy-enthalpy compensation with a larger change in the enthalpy term compared to the entropy term.

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.