Isolation of an individual allosteric interaction in tetrameric phosphofructokinase from Bacillus stearothermophilus.
Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a model allosteric enzyme system in which the interactions between substrates and allosteric effectors have been extensively studied. However, the oligomeric nature of BsPFK has made it difficult to determine the molecular basis of the allosteric regulation because of the multitude of different types of heterotropic and homotropic interactions that are possible between the four active sites and four allosteric sites in the native tetramer. In an attempt to alleviate the complexity of the system and thereby allow the quantitation of a single interaction between one active site and one allosteric site, site-directed mutagenesis has been coupled with a hybrid-forming scheme to create and isolate a tetramer of BsPFK in which only a single active site and a single allosteric site are capable of binding their respective ligands with high (i.e., near wild type) affinity. Characterization of this single allosteric interaction indicates that the free energy involved in the inhibition by the allosteric effector phosphoenolpyruvate (PEP) is 1.48 +/- 0.15 kcal/mol compared to the 3.58 +/- 0.02 kcal/mol measured for the enzyme.
Kimmel
J L
JL
Department of Biochemistry and Biophysics and Center for Advanced Biomolecular Research, Texas A&M University, 2128 TAMU, College Station, Texas 77843-2128, USA.
Reinhart
G D
GD
D000494:Allosteric Regulation; D000495:Allosteric Site; D019943:Amino Acid Substitution; D001665:Binding Sites; D005636:Fructosephosphates / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics
D005636:Fructosephosphates; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C027618:fructose-6-phosphate; D010728:Phosphoenolpyruvate; D010732:Phosphofructokinase-1