Tian, Xinxin (2016-12). Probing the Details of the Allosteric Inhibition in Phosphofructokinase from Thermus thermophilus. Doctoral Dissertation. Thesis uri icon

abstract

  • The enzyme phosphofructokinase (PFK,) catalyzes the phosphorylation of fructose-6-phosphate in the glycolysis pathway. Phosphoenolpyruvate (PEP) allosterically inhibits the binding of the substrate fructose-6-phosphate (Fru-6-P) in phosphofructokinase from Thermus thermophilus (TtPFK). The main goal of this study is to have a better understanding about how this allosteric inhibition signal is transmitted and propagated throughout the enzyme. TtPFK is homotetramer with four active sites and four allosteric sites. There are multiple heterotropic interactions between active sites and allosteric sites. The first part of this dissertation is to isolate the four unique heterotropic inhibition interactions in wild type TtPFK. Our data shows the contribution of the four interactions are not the same, and are additive. This result suggests that the traditional two state model, either the concerted or sequential model, is not sufficient to explain the allosteric regulation in TtPFK. Also, the relative contribution of the four interactions in TtPFK is different from BsPFK and EcPFK. The allosteric coupling between Fru-6-P and PEP in TtPFK is much weaker than BsPFK. N59D/A158T/S215H substitutions increase the coupling free energy of TtPFK similar to BsPFK. The second part of this dissertation is to isolate the four interactions in TtPFK N59D/A158T/S215H to see how the substitutions affect the coupling free energy in each of the four interactions. Our data shows that the substitutions of N59D/A158T/S215H can enhance all of the four interactions, but to different extents. 32 A interaction exhibits the biggest increase in coupling free energy and this big increase makes it the second biggest contribution to TtPFK N59D/A158T/S215H. The coupling free energy in the isolated interactions sums to 69.5% ? 1.5% of the total coupling energy in the native tetramer. The discrepancy is likely due to the mutated residues not all interacting within a single subunit. The third part of this dissertation is to use fluorescence phasor to describe the four species, Apo-TtPFK, TtPFK-Fru-6-P, PEP-TtPFK, and PEP-TtPFK-Fru-6-P, involved in the allosteric coupling between Fru-6-P and PEP. TtPFK has a smaller allosteric coupling between PEP and Fru-6-P as compared to other prokaryotic PFKs which makes it easier to form ternary complex. Unique ternary complexes can be detected at specific positions. Our results suggest that residues F140, L313, F165 and V243 may be in an area important for the propagation and transmission of allosteric information in TtPFK. These four residues are in a region that can detect the structural conflict between Fru-6-P binding and PEP binding.
  • The enzyme phosphofructokinase (PFK,) catalyzes the phosphorylation of fructose-6-phosphate in the glycolysis pathway. Phosphoenolpyruvate (PEP) allosterically inhibits the binding of the substrate fructose-6-phosphate (Fru-6-P) in phosphofructokinase from Thermus thermophilus (TtPFK). The main goal of this study is to have a better understanding about how this allosteric inhibition signal is transmitted and propagated throughout the enzyme.

    TtPFK is homotetramer with four active sites and four allosteric sites. There are multiple heterotropic interactions between active sites and allosteric sites. The first part of this dissertation is to isolate the four unique heterotropic inhibition interactions in wild type TtPFK. Our data shows the contribution of the four interactions are not the same, and are additive. This result suggests that the traditional two state model, either the concerted or sequential model, is not sufficient to explain the allosteric regulation in TtPFK. Also, the relative contribution of the four interactions in TtPFK is different from BsPFK and EcPFK.

    The allosteric coupling between Fru-6-P and PEP in TtPFK is much weaker than BsPFK. N59D/A158T/S215H substitutions increase the coupling free energy of TtPFK similar to BsPFK. The second part of this dissertation is to isolate the four interactions in TtPFK N59D/A158T/S215H to see how the substitutions affect the coupling free energy in each of the four interactions. Our data shows that the substitutions of N59D/A158T/S215H can enhance all of the four interactions, but to different extents. 32 A interaction exhibits the biggest increase in coupling free energy and this big increase makes it the second biggest contribution to TtPFK N59D/A158T/S215H. The coupling free energy in the isolated interactions sums to 69.5% ? 1.5% of the total coupling energy in the native tetramer. The discrepancy is likely due to the mutated residues not all interacting within a single subunit.

    The third part of this dissertation is to use fluorescence phasor to describe the four species, Apo-TtPFK, TtPFK-Fru-6-P, PEP-TtPFK, and PEP-TtPFK-Fru-6-P, involved in the allosteric coupling between Fru-6-P and PEP. TtPFK has a smaller allosteric coupling between PEP and Fru-6-P as compared to other prokaryotic PFKs which makes it easier to form ternary complex. Unique ternary complexes can be detected at specific positions. Our results suggest that residues F140, L313, F165 and V243 may be in an area important for the propagation and transmission of allosteric information in TtPFK. These four residues are in a region that can detect the structural conflict between Fru-6-P binding and PEP binding.

publication date

  • December 2016