The number of allosteric sites and active sites in phosphofructokinase from Bacillus stearothermophilus create an intricate network of communication within the enzyme. With thermodynamic linkage analysis, the overall allosteric communication can be quantified. This value, however, represents an average contribution for all the interactions involved. The recent development of a hybrid strategy has allowed for the quantification of single interactions, both heterotropic and homotropic. Focusing on the heterotropic interactions whose inhibition is entropy-driven, residues and regions within the enzyme can now be identified to further characterize each specific interaction using the hybrid strategy. Among the many components of entropy, the hybrid strategy has now allowed for the strategic placement of a reporter of side chain dynamics to identify conformational differences between the four ligand bound enzyme species of a single heterotropic interaction. In this study, a combination of these approaches was used in the methodology including constructing hybrids to isolate a single heterotropic interaction along with single tryptophan reporter. Site directed mutagenesis combined with the hybrid strategy was also implemented to directly assess the role of a single residue in the communication path of a single interaction. The region surrounding the allosteric site with the nearest active site has been implicated to be significant in transmitting the allosteric signal. In addition two single residues, T158 and D59, within this region have been identified to potentially contribute to the inhibition of this same interaction. An additional residue, G184, located outside this local region has also been identified as possibly having a significant role in the transmission of the inhibitory signal of a unique heterotropic interaction. The implications of this study have led to the initial identification of residues involved in the 22A route of allosteric communication of a single active site and allosteric site. This allosteric communication occurs to allow the enzyme to compensate for the binding of both ligands. With the location of these residues implicated to be involved in the communication of this isolated interaction, this compensation is not contained within a confined region but is however felt throughout the single subunit.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/148105 |
| Date | 14 March 2013 |
| Creators | Perez, Stephanie |
| Contributors | Reinhart, Gregory D |
| Source Sets | Texas A and M University |
| Detected Language | English |
| Type | Thesis, text |
| Format | application/pdf |
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