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

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Title: Kinetics and dynamics study on the allosteric pathway of phosphofructokinase from Escherichia coli
Author: Tie, Cuijuan
Abstract: 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 .
URI: http : / /hdl .handle .net /1969 .1 /85900
Date: 2008-10-10

Citation

Kinetics and dynamics study on the allosteric pathway of phosphofructokinase from Escherichia coli. Available electronically from http : / /hdl .handle .net /1969 .1 /85900 .

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