Disentangling the Web of Allosteric Communication in a Homotetramer:  Heterotropic Inhibition of Phosphofructokinase from Bacillus stearothermophilus

Abstract

A strategy for isolating each of the four potentially unique heterotropic pairwise allosteric interactions that exist in the homotetramer phosphofructokinase from Bacillus stearothermophilus is described. The strategy involves the construction of hybrid tetramers containing one wild-type subunit and three mutant subunits that have been modified to block binding of both the substrate, fructose 6-phosphate (Fru-6-P), and the allosteric inhibitor, phospho(enol)pyruvate (PEP). Each type of binding site occurs at a subunit interface, and mutations on either side of the interface have been identified that will greatly diminish binding at the respective site. Consequently, four different types of mutant subunits have been created, each containing a different active site and allosteric site modification. The corresponding 1:3 hybrids isolate a different pair of unmodified substrate and allosteric sites with a unique structural disposition located 22, 30, 32, and 45 A apart, respectively. The allosteric inhibition exhibited by the unmodified sites in each of these four hybrids has been quantitatively evaluated in terms of a coupling free energy. Each of the coupling free energies is unique in magnitude, and their relative magnitudes vary with pH. Importantly, the sum of these coupling free energies at each pH is equal to the total heterotropic coupling free energy associated with the tetrameric enzyme. The latter quantity was assessed from the overall inhibition of a control hybrid that removed the homotropic interactions in PEP binding. The results do not agree with either the concerted or sequential models that are often invoked to explain allosteric behavior in oligomeric enzymes.

Redefining the Role of the Quaternary Shift in Bacillus stearothermophilus Phosphofructokinase

“…In contrast, the current study, as well as previous studies conducted with BsPFK (9, 10, 27, 28), provides evidence that is difficult to reconcile with this proposal. The crystal structure of the apo D12A BsPFK indicates that the enzyme has undergone the quaternary shift previously associated with the binding of the inhibitor; however, analysis of its coupling parameters shows that the mutant enzyme exhibits an extent of inhibition by PEP that is nearly equivalent to that of wild-type.…”

“…R252 forms intra-subunit interactions with both D12 and Fru-6-P when it is bound. Previous comparisons of the mutant enzymes R252A and R252A/D12A to each other and wild-type reveal that the introduction of the D12A mutation to the R252A variant did not substantially alter the maximal specific activity of the enzyme (10, 13). The D12A enzyme forms stable homotetramers, both in the presence and absence of PEP.…”

“…Previous work performed by Ortigosa, et al noted the significance of D12 when constructing certain hybrid forms of BsPFK (10). In that study, a D12A mutation was made in concert with other mutations in order to allow the proper exchange of subunits when making hybrids.…”

Directed evolution of protein switches and their application to the creation of ligand-binding proteins

“…Enzymes with allosteric effects as large as two orders of magnitude are not common, but they do exist. For example, the binding of phosphonenolpyruvate to Bacillus stearothermophilus phosphofructokinase decreases its affinity for its substrate by Ϸ100-fold (33).…”

The effect of introducing small cavities on the allosteric inhibition of phosphofructokinase from Bacillus stearothermophilus

“…The allosteric coupling between these sites has been previously shown to make the largest contribution to the overall heterotropic allosteric coupling free energy in BsPFK [18]. We recently probed the role of the residues in this region in the allosteric interaction by examining the influence of 3 chimeric substitutions on the allosteric coupling in PFK from the extreme thermophile Thermus thermophilus (TtPFK) [20].…”