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

Ortigosa, Allison D.; Kimmel, Jennifer; Reinhart, Gregory D.

doi:10.1021/bi035077p

Cited by 21 publications

(36 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

Section: Discussioncontrasting

confidence: 99%

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Redefining the Role of the Quaternary Shift in Bacillus stearothermophilus Phosphofructokinase

et al. 2013

Self Cite

View full text Add to dashboard Cite

Bacillus stearothermophilus PFK (BsPFK) is a homotetramer that is allosterically inhibited by phosphoenolpyruvate (PEP), which binds along one dimer-dimer interface. The substrate, fructose 6-phosphate (Fru-6-P), binds along the other dimer-dimer interface. Evans et al., observed that the inhibitor, phosphoglycolate, bound structure, when compared to the substrate and activator bound structure of wild-type BsPFK, exhibits a 7° rotation about the substrate-binding interface, termed the quaternary shift [Schirmer, T., and Evans, P. R. (1990) Nature 343, 140-145]. We report that the variant D12A BsPFK exhibits a 100-fold increase in the binding affinity for PEP, a 50-fold decrease in the binding affinity for Fru-6-P, but an inhibitory coupling comparable to wild type. Crystal structures of the apo and PEP bound forms of D12A BsPFK have been determined (Protein Data Bank ID codes 4I36 and 4I7E, respectively), and both indicate a shifted structure similar to the inhibitor-bound structure of wild type. D12 does not directly bind to either substrate or inhibitor and is located along the substrate-binding interface. A conserved hydrogen bond between D12 and T156 forms across the substrate-binding subunit-subunit interface in the substrate-bound form of BsPFK. The variant T156A BsPFK, when compared to wild-type, shows a 30-fold increase in PEP binding affinity, a 17-fold decrease in Fru-6-P binding affinity, and an estimated coupling that is also approximately equal to wild-type. In addition, the T156A BsPFK crystal structure bound to PEP is reported (Protein Data Bank ID code 4I4I), and it exhibits a shifted structure similar to D12A BsPFK and the inhibitor-bound structure of wild type. The results suggest that main role of the quaternary shift may be to influence ligand binding and not to cause the heterotropic allosteric inhibition per se.

show abstract

Section: Discussioncontrasting

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Redefining the Role of the Quaternary Shift in Bacillus stearothermophilus Phosphofructokinase

et al. 2013

Self Cite

View full text Add to dashboard Cite

show abstract

“…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).…”

Section: Discussionmentioning

confidence: 99%

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

Guntas

Mansell

Kim

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

199

246

View full text Add to dashboard Cite

We describe an iterative approach for creating protein switches involving the in vitro recombination of two nonhomologous genes. We demonstrate this approach by recombining the genes coding for TEM1 ␤-lactamase (BLA) and the Escherichia coli maltose binding protein (MBP) to create a family of MBP-BLA hybrids in which maltose is a positive or negative effector of ␤-lactam hydrolysis. Some of these MBP-BLA switches were effectively ''on-off'' in nature, with maltose altering catalytic activity by as much as 600-fold. The ability of these switches to confer an effector-dependent growth͞no growth phenotype to E. coli cells was exploited to rapidly identify, from a library of 4 ؋ 10 6 variants, MBP-BLA switch variants that respond to sucrose as the effector. The transplantation of these mutations into wild-type MBP converted MBP into a ''sucrose-binding protein,'' illustrating the switches potential as a tool to rapidly identify ligand-binding proteins.allostery ͉ ␤-lactamase ͉ maltose binding protein R egulation of protein activity is fundamental to cellular function.One of the mechanisms a cell uses to modulate the level of protein activity is regulation of the amount of a protein present in a cell. Accordingly, many strategies for engineering control of cellular protein activity have focused on modulation of protein production and degradation, often through small-moleculedependent switches that regulate transcription, translation, localization, degradation, or protein splicing (1) or through the engineering of artificial gene-regulatory networks (2). The key strength of many of these approaches is that they are easily generalized. For example, a switch that regulates transcription of one gene can easily be adapted to regulate transcription of an arbitrary gene. However, the significant limitation of these approaches is the slow dynamics stemming from the indirect nature of the regulation (i.e., protein activity is regulated by controlling the amount of protein and not by regulating the protein's specific activity directly). In addition, modulation is only feasible in the context of the cell and cannot be easily transferred to an in vitro setting.A more satisfying approach is to modulate the protein activity directly, but this presents a considerable design problem. Inhibitors, if they can be found, can only can be used to down-regulate activity, and such a strategy suffers from the fact that one is not free to choose the signal that modulates the activity: The signal must be an inhibitor of the protein. One clever way around this limitation is to engineer the inhibitor such that a third molecule can regulate the inhibitor's affinity for the regulated protein, as was demonstrated for RNA aptamer inhibitors that could be regulated by an organic small molecule (3). Natural allosteric proteins solve this problem by having spatially distinct regulatory and active sites. Ligand binding or covalent modifications at one site affects the output function at a distant site through a conformational change. This mechanism has ...

show abstract

“…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].…”

Section: Introductionmentioning

confidence: 99%

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

Whitaker

Reinhart

2016

Archives of Biochemistry and Biophysics

Self Cite

View full text Add to dashboard Cite

The allosteric coupling free energy between ligands fructose-6-phosphate (Fru-6-P) and phospho(enol)pyruvate (PEP)1 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.

show abstract

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

Cited by 21 publications

References 24 publications

Redefining the Role of the Quaternary Shift in Bacillus stearothermophilus Phosphofructokinase

Redefining the Role of the Quaternary Shift in Bacillus stearothermophilus Phosphofructokinase

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

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

Contact Info

Product

Resources

About