The full text of this item is not available at this time because the student has placed this item under an embargo for a period of time. The Libraries are not authorized to provide a copy of this work during the embargo period, even for Texas A&M users with NetID.
Expression, Purification, and Kinetic Characterization of Human Liver Phosphofructokinase
MetadataShow full item record
Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F16BP) in a MgATP dependent reaction. This reaction represents the first committed step of the glycolytic pathway and as such, plays an important role in metabolism. In liver, this regulation is especially interesting, as hepatocytes can alternatively undergo gluconeogenesis or glycolysis. While PFKs from the livers of several mammalian species have been characterized, human liver PFK has not been thoroughly examined. The gene encoding human liver PFK was cloned into an expression vector utilizing the tac promoter. Human liver PFK was expressed in an Escherichia coli strain, RL257a, which contains no native PFK. The protein has been purified to a specific activity of 130 U/mg through a combination of heat denaturation, ammonium sulfate precipitation, anion exchange chromatography, and gel filtration chromatography. Human liver PFK is inhibited by MgATP with a coupling free energy (ΔGax) of 3.0 kcal/mol, as compared to rat liver PFK (ΔGax 2.3 kcal/mol), a well characterized mammalian PFK. Additionally, human liver PFK demonstrates greater than 10-fold increase in F6P affinity when the pH is increased as demonstrated by the dissociation constants at pH 6 (Kia 0.70) and pH 9 (Kia 0.032). At the cellular concentration of 3 mM MgATP, F16BP activates the enzyme (ΔGax -0.69 kcal/mol), while citrate inhibits the enzyme (ΔGax 0.62 kcal/mol). The additional activators ammonium sulfate (ΔGax -2.0 kcal/mol) and AMP also impact the activity of HLPFK. These results suggest that the human liver enzyme is less sensitive to allosteric effectors than the rat liver enzyme, with the possible exception of MgATP.
Tindall, Amanda Jean (2016). Expression, Purification, and Kinetic Characterization of Human Liver Phosphofructokinase. Master's thesis, Texas A & M University. Available electronically from