Use of 2-Photon Fluorescence Correlation Spectroscopy to Investigate Rat Liver Phosphofructokinase Self-Association

Abstract

Phosphofructokinase (PFK) catalyzes the first committed step of glycolysis. The smallest active oligomer of PFK from rat liver (RLPFK) is a tetramer, however previous studies have demonstrated that at a physiological enzyme concentrations, fructose 6-phosphate (F6P) stabilizes species much larger than a tetramer. Using a Weber linkage argument, we propose that a highly associated species would demonstrate a higher affinity for F6P. 2-photon fluorescence correlation spectroscopy was used to quantify the oligomeric state of RLPFK in the presence of either F6P or MgATP. 11.6μM RLPFK labeled with fluorescein isothiocyanate, initially in the presence of F6P, was diluted to various concentrations from 116 nM to 1.5 nM into buffer with various concentrations, ranging from 3.2 mM to 0.2 mM, of either F6P and observed for 4 hours after dilution. In the presence of MgATP, 116nM RLPFK exists as a stable species with an apparent diffusion coefficient of 24μm2/sec, consistent with the value predicted for a tetramer. Diluting RLPFK to a concentration of 58nM or below in the presence of MgATP resulted in some dissociation into species smaller than the tetramer. The tetramer was stabilized by high [MgATP]. Dilution of RLPFK to 116nM in the presence of F6P gave an apparent diffusion coefficient of 8μM2/sec indicating a species much larger than a tetramer. This large species was stable over four hours in the presence of 3.2 to 0.8 mM F6P. At F6P concentrations of 0.4 mM and lower, RLPFK slowly dissociated into smaller species with an apparent diffusion coefficient of 16μM2/sec. The rate and extent of dissociation was dependent on both [RLPFK] and [F6P]. At low concentration of RLPFK and F6P the enzyme dissociates into species smaller than a tetramer. Funding: NIH-GM33216, NIH-CBI and Welch-A1543.