title: A proteolyzed derivative of Escherichia coli phosphofructokinase is no longer sensitive to allosteric effectors and still shows cooperativity in substrate binding.
abstract: Limited proteolysis of Escherichia coli phosphofructokinase by subtilisin yields a homogeneous derivative. This proteolyzed protein is composed of four polypeptide chains, with a molecular weight of 32 000 as compared to 37 000 for the original enzyme. Removal on each chain of about 5 kdaltons maintains the enzymatic activity and does not change the apparent affinity for the substrates ATP and fructose 6-phosphate. Limited proteolysis, however, affects the cooperativity of fructose 6-phosphate binding: the Hill coefficient is reduced from almost 4 in the native enzyme to only 2 in its proteolyzed derivative. Also, the proteolyzed protein is no longer sensitive to allosteric effectors, activator, or inhibitor. These changes in regulatory properties upon proteolysis are apparently due to the destruction of the effector binding site. The allosteric effector GDP protects phospho-fructokinase against proteolysis and irreversible thermal inactivation; GDP is, however, inefficient in protecting the proteolyzed protein against thermal denaturation. These results suggest that phosphofructokinase may function as a dimer of dimers, in which homotropic and heterotropic allosteric effects are not mediated by the same sets of quaternary interactions.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005636:Fructosephosphates / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006358:Hot Temperature; D046911:Macromolecular Substances; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011489:Protein Denaturation; D013381:Subtilisins / Q000378:metabolism
chemical_list: D005636:Fructosephosphates; D046911:Macromolecular Substances; D006153:Guanosine Diphosphate; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D013381:Subtilisins