Kinetic Characterization of Human Liver Phosphofructokinase

Abstract

Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate in an ATP 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 cell line, RL257a, which contains no native PFK. The protein has been purified to a specific activity of 105 U/mg through a combination of heat denaturation, ammonium sulfate precipitation, anion exchange chromatography, and gel filtration chromatography. Human liver PFK is inhibited by ATP with a coupling free energy (ΔGax) of 3.0 kcal/mol, as compared to rat liver PFK (ΔGax 4.1 kcal/mol), a well characterized mammalian PFK. Additionally, human liver PFK demonstrates a 10-fold increase in F6P affinity when the pH is increased as demonstrated by the dissociation constants at pH 7 (Kia 0.26) and pH 9 (Kia 0.032). Ammonium sulfate activates the enzyme (ΔGax −1.9 kcal/mol), while citrate inhibits the enzyme (ΔGax 0.89 kcal/mol). This work seeks to quantify the kinetic and allosteric behaviors of human liver PFK and compare the magnitude of the effects between rat and human liver PFKs and is supported by the NIH, Grant #GM033216.