Kinetics and dynamics study on the allosteric pathway of phosphofructokinase from Escherichia coli

Phosphofructokinase from Escherichia coli (EcPFK) is allosterically regulated by MgADP and phosphoenolpyruvate (PEP), which act to activate or inhibit, respectively, by changing the substrate (Fru-6-P) affinity of the enzyme. Both ligands bind to the same allosteric site in EcPFK. Therefore, the questions we want to address are how these two molecules regulate EcPFK and how the allosteric signal is propagated throughout the enzyme. EcPFK has 28 potential site-site interactions. These interactions in turn derive from multiple copies of 6 potentially unique homotropic interactions and 4 potentially unique heterotropic interactions. Making hybrid tetramer of EcPFK is used to isolate a single heterotropic interaction. To improve the yield of the 1:3 hybrid, the in vivo hybrid formation method was developed. Four heterotropic interactions were isolated by this manner and re-evaluated. The same kinetics characteristics were obtained for each 1:3 hybrid from both the in vivo and in vitro method. To address the question of how the allosteric signal is transmitted throughout EcPFK, we identified residues (G184, Asp59 and S157) that are important for the allosteric regulation for both PEP inhibition and MgADP activation. The impact of each mutation on individual interaction is unique and also suggests that the structural basis for PEP inhibition is different from that for MgADP activation. Most importantly, since the sum of each heterotropic interaction with a modification in only one subunit is equal to the total heterotropic interaction with a modification in all four subunits, this result indicates that the heterotropic allosteric signal transmission is realized in a single subunit. The 23? heterotropic interaction, which contributes the most to the PEP inhibition, was chosen to study the dynamic properties. Fluorescence was used to study the dynamic perturbations of the 23? interaction upon ligand binding. Taking advantage of the hybrid formation strategy and the tryptophan-shift mutagenesis method, a tryptophan residue can be placed at different individual locations throughout the native subunit containing the 23? heterotropic interaction. The steady-state anisotropy and lifetime measurement at each tryptophan position indicate that the 23? allosteric interaction involves the perturbation of side-chain dynamics both near and quite far away from the respective ligand binding sites.