Isolation of a single activating allosteric interaction in phosphofructokinase from Escherichia coli.
Escherichia coli phosphofructokinase 1 (EcPFK) is a homotetramer with four active and four allosteric sites. Understanding of the structural basis of allosteric activation of EcPFK by MgADP is complicated by the multiplicity of binding sites. To isolate a single heterotropic allosteric interaction, hybrid tetramers were formed between wild-type and mutant EcPFK subunits in which the binding sites of the mutant subunits have decreased affinity for their respective ligands. The 1:3 (wild-type:mutant) hybrid that contained only one native active site and one native allosteric site was isolated. The affinity for the substrate fructose-6-phosphate (Fru-6-P) of a single wild-type active site is greatly decreased over that displayed by the wild-type tetramer due to the lack of homotropic activation. The free energy of activation by MgADP for this heterotropic interaction is -0.58 kcal/mol at 8.5 degrees C. This compares to -2.87 kcal/mol for a hybrid with no homotropic coupling but all four unique heterotropic interactions. Therefore, the isolated interaction contributes 20% of the total heterotropic coupling. By comparison, wild-type EcPFK exhibits a coupling free energy between Fru-6-P and MgADP of -1.56 kcal/mol under these conditions, indicating that the effects of MgADP are diminished by a homotropic activation equal to -1.3 kcal/mol. These data are not consistent with a concerted allosteric mechanism.
Fenton
Aron W
AW
Department of Biochemistry and Biophysics and the Center for Advanced Biomolecular Research, Texas A&M University, 2128 TAMU, College Station, Texas 77843-2128, USA.
Reinhart
Gregory D
GD
D000244:Adenosine Diphosphate / Q000737:chemistry; D000495:Allosteric Site / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D005636:Fructosephosphates / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity / Q000235:genetics
D029968:Escherichia coli Proteins; D005636:Fructosephosphates; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D000244:Adenosine Diphosphate; C027618:fructose-6-phosphate; D010732:Phosphofructokinase-1