Exploration of the Interplay Between Allosteric Interactions of Phosphofructokinase From Rat Liver

Abstract

The key metabolic enzyme phosphofructokinase (PFK) catalyzes the phosphorylation of fructose-6-phosphate (Fru-6-P) by MgATP in the first committed step of glycolysis. PFK’s phosphorylation activity is tightly regulated by allosteric effectors who act by either increasing or decreasing its affinity to substrate Fru-6-P. Liver PFK from rats (RLPFK) is allosterically regulated by several metabolic byproducts including MgATP, citrate and AMP. Despite the importance of precise regulation of this enzyme, the interplay between the different allosteric effectors has not been thoroughly characterized. Presented here are the effects of MgATP on the ability of allosteric activator (AMP) or inhibitor (citrate) to modulate the activity of RLPFK. We see that MgATP dramatically decreases the ability of both AMP and citrate to allosterically regulate RLPFK. RLPFK has additionally been implemented to be subjected to activation through a novel self-association mechanism. This is difficult to prove unequivocally as the kinetic assays used to measure enzyme activity are performed (by necessity) at concentrations thought to be too low for self-association to occur. It has been demonstrated that at a physiological enzyme concentration Fru-6-P promotes self-association. We propose that if Fru-6-P promotes the formation self-associated species, then self-association must increase its affinity for Fru-6-P. Utilizing Fluorescence Correlation Spectroscopy (FCS) we measured the size of RLPFK particles at concentrations that span the gap between those used for kinetic assays and those representing physiological conditions. Additionally, we varied the concentration of ligand (either MgATP or Fru-6-P). Our data demonstrate that Fru-6-P acts cooperatively with concentration of RLPFK to increase the extent of self-association, whereas no self-association occurs in the presence of MgATP. These FCS data provide exciting insight into the role of activation by self-association and provides the foundation for which future experiments can expound.