Spectroscopic studies on effector-induced and substrate-induced conformation changes of phosphofructokinase.
The interaction of phosphofructokinase with NH4+, AMP, ATP, citrate, MgATP or fructose 6-phosphate, and in part with their mixtures forming either binary or ternary complexes has been studied by means of ultraviolet difference spectroscopy and circular dichroism spectroscopy in the wavelength range 265-300 nm with the aim of characterizing the conformational corollaries of the ligand effects on phosphofructokinase. The positive as well as the negative effectors change phosphofructokinase conformation in different ways, not easily interpretable in terms of one active and one inactive enzyme conformation. The spectroscopic equivalents of phosphofructokinase conformation changes resulting from catalytic activity are similar to those produced by the reaction products. The ligand concentration-dependent changes of absorption differences in the tryptophyl, tyrosyl and phenylalanyl region parallel each other, i.e. the interactions of the ligands with phosphofructokinase are not confined to specific aromatic side chains, but involve conformation changes of the large domains of the protein. ATP affinity to the enzyme shows temperature-dependent biphasic changes so that ATP binding appears to be either an entropy-driven or enthalpy-driven process. The dissociation constants of the ligands derived from spectroscopic titration of binary complex formation are comparable to those calculated from kinetic experiments. MgATP and fructose 6-phosphate each alone change phosphofructokinase conformation by binary complex formation in keeping with a random order of reaction sequence.
D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000641:Ammonia; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D002951:Citrates; D005636:Fructosephosphates; D007700:Kinetics; D008274:Magnesium; D009132:Muscles / Q000201:enzymology; D010732:Phosphofructokinase-1; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D002951:Citrates; D005636:Fructosephosphates; D000249:Adenosine Monophosphate; D000641:Ammonia; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D008274:Magnesium