Browsing by Subject "phosphofructokinase"
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Item Illuminating the Heterotropic Communication of the Pair-wise Interactions in Phosphofructokinase from Bacillus stearothermophilus(2012-10-17) Perez, StephanieThe 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.Item Pathway to allostery: differential routes for allosteric communication in phosphofructokinase from Escherichia coli(Texas A&M University, 2005-02-17) Paricharttanakul, Nilubol MoniquePhosphofructokinase from Escherichia coli (EcPFK) is allosterically regulated by MgADP and phospho(enol)pyruvate (PEP). Both molecules compete for binding to the same allosteric site, however, MgADP activates and PEP inhibits the binding of fructose-6-phosphate (F6P) to the active site. The mode by which this enzyme can differentiate between the two ligands and cause the appropriate response is important for the understanding of the basis of allosteric regulation. We studied the interactions between an active site and an allosteric site (heterotropic interactions) within the protein, and found that each of the four unique heterotropic interactions is unique and the magnitudes of the coupling free energies for MgADP activation sum up to 100% that of wildtype EcPFK without homotropic cooperativity in F6P binding. We took on the kinetic and structural characterization of phosphofructokinase from Lactobacillus bulgaricus (LbPFK) to reveal an enzyme that exhibits allosteric properties in spite of previous kinetic studies performed by Le Bras et al. (1991). We have identified residues in EcPFK (Asp59, Gly184 and Asp273), which are important for the allosteric responses to both MgADP and PEP. Interestingly, Lys214 is only important in PEP inhibition and not MgADP activation. We can also differentially disrupt the MgADP heterotropic interactions with the introduction of G184C within the protein. These results suggest that there are different pathways for allosteric communication within the enzyme: different paths for MgADP activation and PEP inhibition, and different paths for each heterotropic interaction with Gly184 being important for the 33? MgADP heterotropic interaction.Item Resolution of the pair-wise allosteric interactions found in phosphofructokinase from Bacillus stearothermophilus(Texas A&M University, 2004-09-30) Ortigosa, Allison DawnPhosphofructokinase from Bacillus stearothermophilus (BsPFK) is a homotetrameric enzyme with an average of one active site and one allosteric site per subunit. BsPFK is inhibited by phosphoenolpyruvate (PEP) and how this inhibitory signal is propagated throughout the enzyme is the main question we address through this investigation. By possessing a total of eight binding sites, a potential for twenty-eight total pair-wise allosteric interactions result within BsPFK, ten of which are unique. Of these ten interactions, four are heterotropic interactions, or interactions between unlike binding sites, while the remaining six interactions are homotropic interactions, or interactions between like binding sites. Thus, to address the question of how BsPFK is inhibited by PEP, each of these ten interactions needs to be quantified and their roles in the inhibition process assessed. In order to quantify the roles of the 10 allosteric interactions, we created, purified and characterized several different hybrid enzymes by using site-directed mutagenesis to reduce the number of native active sites and native allosteric sites to permit the isolation of specific allosteric interaction(s). Through the creation and isolation of 1:3 hybrid enzymes, in which one native active site and one native allosteric site remain, each of the four heterotropic interactions were characterized. Moreover, through the creation and isolation of the 2:2 hybrid enzymes, in which two native active sites and two native allosteric sites remain, characterization of the remaining six homotropic interactions was performed. Utilizing a linked function approach to quantify the heterotropic and homotropic effects for each hybrid enzyme, we determined that 5 to 6 of the ten pair-wise allosteric interactions found in BsPFK are involved in the inhibition process depending upon pH. More importantly however, our data provides definitive results that the traditional two-state models used to describe an allosteric effect are not sufficient to describe the allosteric effect measured for BsPFK. Rather, our results show that the linked function approach is a more appropriate way to unambiguously measure the nature and magnitude of an allosteric effect. Moreover, this approach can also be used to explain the allosteric behavior of a dimeric enzyme.