Use of 2-Photon Fluorescence Correlation Spectroscopy and Electron Microscopy to Ellucidate the Dependence of RLPFK Self Association on Ligand Concentration

Abstract

Phosphofructokinase (PFK) catalyzes the first committed step of glycolysis. Allosteric regulation of PFK makes its activity, and thus glycolysis, sensitive to intercellular metabolic conditions. Regulation of PFK from liver is complex because of the liver's responsibility to maintain glucose homeostasis, which means that glycolysis and gluconeogenesis must be regulated reciprocally. Allosteric regulation of PFK activity is achieved by modifying the binding affinity of its substrate, fructose 6-phosphate (Fru-6-P). However, in vitro studies performed on rat liver PFK (RLPFK) suggest that at physiological concentrations of Fru-6-P RLPFK activity is negligible even in the presence of known allosteric activators. Additional mechanisms must exist to account for the activity required to support flux through glycolysis. The smallest active oligomer of RLPFK is a tetramer, however fluorescence polarization studies have previously demonstrated that at a physiological enzyme concentration both Fru-6-P and other activators stabilize species much larger than a tetramer. A Weber linkage argument predicts that the highly associated species would demonstrate a higher affinity for Fru-6-P resulting in activation. RLPFK exists as a tetramer in the dilute enzyme concentrations necessary for in vitro activity assays whereas, at physiological concentrations, RLPFK can be highly associated. 2-photon fluorescence correlation spectroscopy (FCS) and electron microscopy have been used to quantify the oligomeric state of RLPFK at high and low enzyme concentrations. FCS performed on alexa-488 labeled RLPFK suggests a complex self-association behavior in the presence of Fru-6-P. Electron microscopy indicates that, in the presence of Fru-6-P, rat liver PFK can form long fibrils that consist of up to 36 PFK tetramers extending over 200 nm in length. These effects are counteracted by MgATP, a known allosteric inhibitor of the enzyme. Funding: NIH-GM33216, NIH-CBI and Welch-A1543