title: Desensitization of muscle phosphofructokinase to ATP inhibition by removal of a carboxyl-terminal heptadecapeptide.
abstract: Brief exposure of rabbit skeletal muscle phosphofructokinase to Staphylococcus aureus V8 protease results in the release from the enzyme of two carboxyl-terminal peptides from the enzyme that together comprise 17 amino acids. The rate of proteolysis was increased in the presence of activators of the enzyme, ammonium sulfate and AMP, and was decreased in the presence of allosteric inhibitors, MgATP and citrate. No change was observed in the maximal velocity of the modified enzyme or in its affinity for substrates when assayed under noninhibitory conditions. Equilibrium binding studies indicated no change in the affinity of the modified enzyme for its allosteric activator, AMP. On the other hand, the proteolyzed enzyme exhibited markedly reduced inhibition by ATP and by citrate. ATP inhibition was observed only at very high concentrations of ATP. Fructose-6-P saturation curves of the modified enzyme were nearly hyperbolic. The interaction coefficient deduced from the slope of a Hill-type plot was 1.2 under conditions that yielded a coefficient of 3.0 with native phosphofructokinase. Binding studies verified a decrease in affinity for ATP for at least one of the ATP binding sites. Because kinetic studies showed no effect on the Km for ATP, it was concluded that the affinity was decreased at the MgATP inhibitory site only. Proteolytic removal of the terminal 8 residues from the enzyme produced no striking change in regulatory properties, thus focusing the critical region to the sequence His-Ala-His-Leu-Glu-His-Ile-Ser-Arg. It is suggested that the three histidine residues clustered in the carboxyl terminus may contribute to the binding of MgATP to the inhibitory site.
mesh_terms: D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D010450:Endopeptidases / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism*; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors*; D011817:Rabbits; D012697:Serine Endopeptidases
chemical_list: D002951:Citrates; D005636:Fructosephosphates; D010446:Peptide Fragments; D019343:Citric Acid; D000249:Adenosine Monophosphate; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D000645:Ammonium Sulfate