Isolation of an individual allosteric interaction in tetrameric phosphofructokinase from Bacillus stearothermophilus.

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a model allosteric enzyme system in which the interactions between substrates and allosteric effectors have been extensively studied. However, the oligomeric nature of BsPFK has made it difficult to determine the molecular basis of the allosteric regulation because of the multitude of different types of heterotropic and homotropic interactions that are possible between the four active sites and four allosteric sites in the native tetramer. In an attempt to alleviate the complexity of the system and thereby allow the quantitation of a single interaction between one active site and one allosteric site, site-directed mutagenesis has been coupled with a hybrid-forming scheme to create and isolate a tetramer of BsPFK in which only a single active site and a single allosteric site are capable of binding their respective ligands with high (i.e., near wild type) affinity. Characterization of this single allosteric interaction indicates that the free energy involved in the inhibition by the allosteric effector phosphoenolpyruvate (PEP) is 1.48 +/- 0.15 kcal/mol compared to the 3.58 +/- 0.02 kcal/mol measured for the enzyme.

Reevaluation of the accepted allosteric mechanism of phosphofructokinase from Bacillus stearothermophilus.

The binding of phosphoenolpyruvate (PEP) to the single allosteric site on phosphofructokinase (EC ) from Bacillus stearothermophilus (BsPFK) diminishes the ability of the enzyme to bind the substrate fructose 6-phosphate (Fru-6-P). Comparisons of crystal structures with either Fru-6-P or phosphoglycolate, an analog of PEP, bound have shown that Arg-162 interacts with the negatively charged Fru-6-P. Upon the binding of phosphoglycolate, Arg-162 is virtually replaced by Glu-161, which introduces a potential coulombic repulsion between enzyme and substrate [Schirmer, T. & Evans, P. R. (1990) Nature (London) 343, 140-145]. It has previously been proposed that this structural transition explains the allosteric inhibition in BsPFK, and this explanation has appeared in textbooks to illustrate how an allosteric ligand can influence substrate binding at a distance. Site-directed mutagenesis has been employed to create three mutants of BsPFK that substitute an alanine residue for Glu-161, Arg-162, or both. The E161A mutation does not affect the inhibition of BsPFK by PEP at 25 degrees C, and while the R162A mutation decreases BsPFK's affinity for Fru-6-P by approximately 30-fold, R162A diminishes the effectiveness of PEP inhibition by only 1/3. Combining E161A and R162A produces behavior comparable to R162A alone. These and other data suggest that the movement of Glu-161 and Arg-162 does not play the central role in producing the allosteric inhibition by PEP as originally envisioned in the Schirmer and Evans mechanism.